Amino acid sequences directed against rank-l and polypeptides comprising the same for the treatment of bone diseases and disorders

ABSTRACT

The present invention relates to amino acid sequences that are directed against RANK-L, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences. The invention also relates to nucleic acids encoding such amino acid sequences and polypeptides; to methods for preparing such amino acid sequences and polypeptides; to host cells expressing or capable of expressing such amino acid sequences or polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such amino acid sequences, polypeptides, nucleic acids and/or host cells; and to uses of such amino acid sequences or polypeptides, nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes.

RELATED APPLICATIONS

This application is a continuation of U.S. provisional application Ser.No. 12/558,032, filed Sep. 11, 2009, which is a continuation-in-part ofinternational application PCT/EP2008/056383, filed May 23, 2008, whichwas published under PCT Article 21(2) in English, which claims thebenefit under 35 U.S.C. §119(e) of U.S. provisional application No.60/939,929, filed May 24, 2007, and of U.S. provisional application No.61/024,256, filed Jan. 29, 2008, each of which is incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to amino acid sequences that are directedagainst (as defined herein) Receptor Activator of Nuclear factor Kappa BLigand (RANK-L, also called tumor necrosis factor-related,activation-induced cytokine (TRANCE), osteoclast differentiation factor(ODF), osteoprotegerin ligand (OPG-L) or tumor necrosis factorsuperfamily member 11 (TNFSF11)), as well as to compounds or constructs,and in particular proteins and polypeptides, that comprise oressentially consist of one or more such amino acid sequences (alsoreferred to herein as “amino acid sequences of the invention”,“compounds of the invention”, and “polypeptides of the invention”,respectively).

The invention also relates to nucleic acids encoding such amino acidsequences and polypeptides (also referred to herein as “nucleic acids ofthe invention” or “nucleotide sequences of the invention”); to methodsfor preparing such amino acid sequences and polypeptides; to host cellsexpressing or capable of expressing such amino acid sequences orpolypeptides; to compositions, and in particular to pharmaceuticalcompositions, that comprise such amino acid sequences, polypeptides,nucleic acids and/or host cells; and to uses of such amino acidsequences or polypeptides, nucleic acids, host cells and/orcompositions, in particular for prophylactic, therapeutic or diagnosticpurposes, such as the prophylactic, therapeutic or diagnostic purposesmentioned herein.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

BACKGROUND OF THE INVENTION

Remodelling (turnover) of bone is the process by which the adultskeleton is continually being resorbed (removed) and formed (replaced).Bone remodeling involves the synthesis of bone matrix by osteoblasts andits resorption by osteoclast cells. Osteoclasts, derived fromhematopoietic cells, are unique forms of tissue macrophages that havethe capacity to resorb bone tissue. Osteoblasts are specializedfibroblasts that have the capacity of secreting bone collagen. There isan exquisite coordination among the activities of these bone cells thatlink the processes of bone formation and bone resorption.

Bone remodelling is controlled by a balance between RANK-L/RANK and theRANK-L decoy receptor OPG. RANK-L and its receptor RANK are essentialfor the development and activation of osteoclasts. OPG, a secretedprotein, is an effective inhibitor of osteoclast maturation andosteoclasts activation. In normal bone homeostasis, RANK-L and OPGparticipate in a cytokine axis that tightly controls the generation ofosteoclasts from monocyte precursors. RANK-L, expressed by osteoblastsand bone marrow stromal cells, binds to its functional receptor, RANK,to stimulate differentiation of osteoclasts from precursor cells and theproliferation and activity of mature osteoclasts. OPG, which isexpressed by osteoblasts, stromal cells, dendritic cells, andmegakaryocytes, limits this process by acting as a soluble decoyreceptor for RANK-L.

The TNF family molecule RANK-L is encoded by a single gene (rankl) athuman chromosome 13q14. RANK-L mRNA is expressed at highest levels inbone and bone marrow, as well as in lymphoid tissues (lymph node,thymus, spleen, fetal liver, and Peyer's patches) (Anderson et al. 1997,Nature 390: 175-179; Wong et al. 1997, J. Biol. Chem. 272: 25190-25194;Lacey et al. 1998, Cell 93: 165-176; Yasuda et al. 1998, Proc. Natl.Acad. Sci. USA 95: 3597-3602). Alternative splicing of RANK-L mRNAallows expression as a type II transmembrane glycoprotein of either 316or 270 amino acids or as a soluble ligand of 243 amino acids (Kong etal. 1999, Nature 397: 315-323; Nagai et al. 2000, Biochem Biophys. Res.Commun 269: 532-536). In addition, RANK-L can be released from itsmembrane bound state by metalloproteinases, including TNF-alphaconvertase (Lum et al. 1999, J. Biol. Chem. 274: 13613-13618). All fourisoforms of RANK-L associate into trimeric molecules capable oftriggering osteoclastogenesis.

RANK (receptor activator of NFkappaB also known as TRANCE-R, ODAR, orTNFRSF11A), expressed on preosteoclastic cells, is the sole receptor onthese cells for RANK-L (Li et al. 2000, Proc. Natl. Acad. Sci. USA 97:1566-1571). RANK activation by RANK-L is followed by its interactionwith TNF receptor-associated (TRAF) family members, activation ofnuclear factor (NF)-kappaB and c-Fos, JNK, c-src, and theserine/threonine kinase Akt/PKB (Anderson et al. 1997, Nature 390:175-179; Hsu et al. 1999, Proc. Acad. Sci. USA 96: 3540-3545).

OPG (osteoprotegerin; “protector of the bone”; also known asosteoclastogenesis inhibitory factor (OCIF)) is a soluble, 110-kDa,disulfide-linked, homodimeric glycoprotein produced and released byactivated osteoblast cells (Simonet et al. 1997, Cell 89: 309-319) withhomology to the TNF receptor family, that functions as a decoy receptorfor RANK-L and competes with RANK for RANK-L binding. Consequently, OPGis an effective inhibitor of osteoclast maturation and osteoclastactivation (Simonet et al. 1997, Cell 89: 309-319; Lacey et al. 1998,Cell 93: 165-176; Kong et al. 1999, Nature 397: 315-323), therebyreducing bone resorption.

A more detailed overview of the OPG/RANK-L/RANK system as the mediatorof bone formation and destruction is presented in Khosla, (2001Endocrinology 142: 5050-5055), Holstead Jones et al. (2002, Ann. Rheum.Dis. 61 (Suppl II): ii32-ii39), Bezerra et al. (2005, Brazilian J. Med.Biol. Res. 38: 161-170) and McClung (2006, Current Osteoporosis Reports4: 28-33).

Several bone disorders occur when there is an imbalance between theresorption and formation components of bone remodeling activity(uncoupling of bone homeostasis). Imbalances between osteoclast andosteoblast activities can arise from a wide variety of hormonal changesor perturbations of inflammatory and growth factors, such as e.g. analtered balance between OPG and RANK-L. When bone resorption is greaterthan bone formation, there is a net loss of bone over time. This caneventually result in low bone mass (osteopenia) or osteoporosis. Whenbone formation exceeds resorption, there is a net increase in bone mass(osteopetrosis).

Excessive bone loss or destruction due to higher RANK-L, lower OPG orboth has been implicated in many disease states, includingpost-menopausal osteoporosis (Eghbali-Fatourechi et al. 2003, Journal ofClinical Investigation 111: 1221-1230; Tsangari et al. 2004, Bone 35:334-342; Abdallah et al. 2005, Calcified Tissue International 76:90-97), primary hyperparathyroidism (Stilgren et al. 2004, Bone 35:256-265; Johnell et al. 2005, Journal of Bone and Mineral Research 20:1185-1194), Paget's disease of bone (Reddy 2004, Journal of CellularBiochemistry 93: 688-696), metastatic bone disease (Brown 2004, CancerTreatment and Research 118: 149-172), myeloma (Okada et al. 2003,Clinical and Experimental Metastasis 20: 639-646), rheumatoid arthritis(Crotti et al. 2002, Annals of the Rheumatic Diseases 61: 1047-1054) andseveral other metabolic or inflammatory bone and joint disorders(Locklin et al. 2001, Bone 28 (Suppl.): S80; Lewiecki 2006, Expert Opin.Biol. Ther. 6: 1041-1050).

Pharmacological agents to decrease risk of fracture have been availablefor more than ten years. Anticatabolic drugs (oestrogens,bisphosphonates, calcitonin and selective oestrogen receptor modulators)decrease bone resorption, while anabolic agents, such as recombinanthuman parathyroid hormone (PTH), increase bone formation and bone size.The bisphosphonate class of drugs is the one most often used for thetreatment of osteoporosis. Although this drug class is generally verysafe, oral dosing is complex and has been associated withgastrointestinal adverse events in a small percentage of clinicalpractice patients. Clinical trials are evaluating increasing intervalsof intravenous bisphosphonate dosing.

The recent discovery of the OPG/RANK-L/RANK system as pivotal regulatoryfactors in the pathogenesis of bone diseases and disorders likeosteoporosis provides unique targets for therapeutic agents. Inlaboratory animals and in humans, administering forms of OPG markedlyinhibited osteoclast activity and improved bone strength (Bekker et al.2001, J. Bone Miner. Res. 16: 348-360; Campagnuolo et al. 2002,Arthritis Rheum. 46: 1926-1936; Bezerra et al. 2005, Brazilian J. Med.Biol. Res. 38: 161-170; McClung 2006, Current Osteoporosis Reports 4:28-33). In early studies in humans, a fully human antibody againstRANK-L (Denosumab) reduced bone turnover and improved bone density (Bodyet al. 2003, Cancer 97: 887-892; Bekker et al. 2004, J. Bone Miner. Res.19: 1059-1066; McClung 2006, Current Osteoporosis Reports 4: 28-33;Lewiecki 2006, Expert Opin. Biol. Ther. 6: 1041-1050; McClung et al.2006, N. Engl. J. Med. 354: 821-831). Such complete antibodies, however,face the drawbacks of full size antibodies such as high productioncosts, low stability, and their large size, which e.g. impedes theiraccess to certain hidden epitopes.

SUMMARY OF THE INVENTION

NANOBODIES (a novel class of proprietary therapeutic proteins derivedfrom heavy-chain variable domains (VHH) that occur naturally in heavychain only immunoglobulins; singular, NANOBODY) are more potent and morestable than conventional four-chain antibodies which leads to (1) lowerdosage forms, less frequent dosage leading to less side effects; and (2)improved stability leading to a broader choice of administration routes,comprising oral or subcutaneous routes and slow-release formulations inaddition to the intravenous route.

Because of their small size, NANOBODIES have the ability to crossmembranes and penetrate into physiological compartments, tissues andorgans not accessible to other, larger polypeptides and proteins.NANOBODIES might, for example, easily penetrate into the bone matrixmaking them suited for the treatment of bone diseases and disorders.

The small size of the NANOBODY also makes them ideally suited for theirengineering into multivalent or multispecific polypeptides. In contrastto full antibodies which can bind to only one subunit of the RANK-Ltrimer, bivalent or trivalent polypeptides (based on respectively two orthree NANOBODIES against RANK-L), will be able to bind on respectively 2or 3 subunits of the trimeric RANK-L molecule and might be advantageousbecause of their higher potency.

The amino acid sequences, polypeptides and compositions of the presentinvention can generally be used to modulate, and in particular inhibitand/or prevent, binding of RANK-L to RANK, and thus to modulate, and inparticular inhibit or prevent, the signalling that is mediated byRANK-L, RANK and/or OPG, to modulate the biological pathways in whichRANK-L and/or RANK are involved, and/or to modulate the biologicalmechanisms, responses and effects associated with such signalling orthese pathways. The binding of RANK-L to RANK and/or the signalling thatis mediated by RANK-L, RANK and/or OPG may be inhibited and/or preventedby at least 1%, preferably at least 5%, such as at least 10% or at least25%, for example by at least 50%, at least 60%, at least 70%, at least80%, or 90% or more, compared to the binding of RANK-L to RANK and/orthe signalling that is mediated by RANK-L, RANK and/or OPG under thesame conditions but without the presence of the amino acid sequence,NANOBODY or polypeptide of the invention.

In another aspect of the present invention, the amino acid sequences,polypeptides and compositions of the present invention can be used tomodulate, and in particular inhibit and/or prevent, binding of RANK-L toOPG, and thus to modulate (inhibit and/or prevent or boost) thesignalling that is mediated by RANK-L, RANK and/or OPG, to modulate thebiological pathways in which RANK-L, RANK and/or OPG are involved,and/or to modulate the biological mechanisms, responses and effectsassociated with such signalling or these pathways. The amino acidsequences, polypeptides and compositions of the present invention may beagonist or antagonist of RANK-L and/or such signalling. They may inhibitRANK/RANK-L mediated signalling in the same way as OPG, or they mayfully or partially prevent OPG from inhibiting RANK/RANK-L mediatedsignalling. The binding of RANK-L to OPG and/or the signalling that ismediated by RANK-L, RANK and/or OPG may be inhibited and/or prevented byat least 1%, preferably at least 5%, such as at least 10% or at least25%, for example by at least 50%, at least 60%, at least 70%, at least80%, or 90% or more, compared to the binding of RANK-L to OPG and/or thesignalling that is mediated by RANK-L, RANK and/or OPG under the sameconditions but without the presence of the amino acid sequence, NANOBODYor polypeptide of the invention.

In another aspect of the present invention, the amino acid sequences,polypeptides and compositions of the invention can be used to modulate(inhibit and/or prevent or boost) the differentiation and/orproliferation of osteoclasts. The differentiation and/or proliferationof osteoclasts may be increased or decreased, respectively, by at least1%, preferably at least 5%, such as at least 10% or at least 25%, forexample by at least 50%, at least 60%, at least 70%, at least 80%, or90% or more, compared to the differentiation and/or proliferation ofosteoclasts under the same conditions but without the presence of theamino acid sequence, NANOBODY or polypeptide of the invention.

In another aspect of the present invention, the amino acid sequences,polypeptides and compositions of the invention can be used to modulatebone remodelling. Bone remodelling may be modulated at least 1%,preferably at least 5%, such as at least 10% or at least 25%, forexample by at least 50%, at least 60%, at least 70%, at least 80%, or90% or more, compared to bone remodelling under the same conditions butwithout the presence of the amino acid sequence, NANOBODY or polypeptideof the invention.

As such, the polypeptides and compositions of the present invention canbe used for the prevention and treatment (as defined herein) of bonediseases and disorders. Generally, “bone diseases and disorders” can bedefined as diseases and disorders that can be prevented and/or treated,respectively, by suitably administering to a subject in need thereof(i.e. having the disease or disorder or at least one symptom thereofand/or at risk of attracting or developing the disease or disorder) ofeither a polypeptide or composition of the invention (and in particular,of a pharmaceutically active amount thereof) and/or of a known activeprinciple active against RANK-L or a biological pathway or mechanism inwhich RANK-L is involved (and in particular, of a pharmaceuticallyactive amount thereof).

Bone diseases and disorders encompass diseases and disorders associatedwith the regulation of bone formation and resorption. Bone diseases anddisorders characterized by a net bone loss (bone resorption exceeds boneformation) are also referred to as osteopenic disorders, includingostopenia, osteoporosis and osteolysis and are characterized byexcessive and/or unwanted signaling mediated by RANK-L. The polypeptidesand compositions of the present invention that modulate, and inparticular inhibit and/or prevent, binding of RANK-L to RANK act asantagonist and will generally be used for the prevention and treatment(as defined herein) of bone diseases and disorders characterized by netbone loss. Also polypeptides and compositions of the present inventionthat modulate, and in particular inhibit and/or prevent, binding ofRANK-L to OPG may act as antagonists and will generally be used for theprevention and treatment (as defined herein) of bone diseases anddisorders characterized by net bone loss.

Bone diseases and disorders characterized by net increase in bone massare referred to as osteopetrosis and are characterized by poor signalingmediated by RANK-L. The polypeptides and compositions of the presentinvention that modulate, and in particular inhibit and/or prevent,binding of RANK-L to OPG may act as agonists and will generally be usedfor the prevention and treatment (as defined herein) of bone diseasesand disorders characterized by net increase in bone mass.

Examples of such bone diseases and disorders will be clear to theskilled person based on the disclosure herein, and for example includethe following diseases and disorders: Osteoporosis (McClung 2006,Current Osteoporosis Reports 4: 28-33), including, but not limited to,primary osteoporosis, endocrine osteoporosis (including, but not limitedto, hyperthyroidism, hyperparathyroidism (Anandarajah and Schwarz 2006,J. Cell Biochem. 97: 226-232), Cushing's syndrome, and acromegaly),hereditary and congenital forms of osteoporosis (including, but notlimited to, osteogenesis imperfecta, homocystinuria, Menkes' syndrome,Riley-Day syndrome), osteoporosis due to immobilization of extremities,glucocorticoid-induced osteoporosis (Locklin et al. 2001, Bone 28(Suppl.): S80; McClung 2006, Current Osteoporosis Reports 4: 28-33;Anandarajah and Schwarz 2006, J. Cell Biochem. 97: 226-232) andpost-menopausal osteoporosis (McClung 2006, Current Osteoporosis Reports4: 28-33); (Juvenile or Familial) Paget's disease (Cundy et al. 2002,Hum. Mol. Genet. 11: 2119-2127; Whyte et al. 2002, J. Bone Miner. Res.17: 26-29; Whyte et al. 2002, N. Engl. J. Med. 347: 175-184;Johnson-Pais et al. 2003, J. Bone Miner Res. 18: 376-380; Anandarajahand Schwarz 2006, J. Cell Biochem. 97: 226-232; Anandarajah and Schwarz2006, J. Cell Biochem. 97: 226-232); Osteomyelitis, i.e., an infectiouslesion in bone, leading to bone loss; Hypercalcemia (Anandarajah andSchwarz 2006, J. Cell Biochem. 97: 226-232), including, but not limitedto, hypercalcemia resulting from solid tumors (including, but notlimited to, breast, lung and kidney) and hematologic malignancies(including, but not limited to, multiple myeloma (Sordillo and Pearse2003, Cancer 97 (3 Suppl): 802-812; Vanderkerken et al. 2003, CancerRes. 63: 287-289), lymphoma and leukemia), idiopathic hypercalcemia, andhypercalcemia associated with hyperthyroidism and renal functiondisorders; Bone loss, including but not limited to, osteopenia followingsurgery, osteopenia induced by steroid administration, osteopeniaassociated with disorders of the small and large intestine, andosteopenia associated with chronic hepatic and renal diseases;Osteonecrosis, i.e., bone cell death, including, but not limited to,osteonecrosis associated with traumatic injury, osteonecrosis associatedwith Gaucher's disease, osteonecrosis associated with sickle cellanemia, osteonecrosis associated with systemic lupus erythematosus,osteonecrosis associated with rheumatoid arthritis, osteonecrosisassociated with periodontal disease, osteonecrosis associated withosteolytic metastasis, and osteonecrosis associated with othercondition; Bone loss associated with arthritic disorders such aspsoriatic arthritis, rheumatoid arthritis, loss of cartilage and jointerosion associated with rheumatoid arthritis (Bezerra et al. 2005,Brazilian Journal of Medical and Biological Research 38: 161-170;Anandarajah and Schwarz 2006, J. Cell Biochem. 97: 226-232); Arthritis(Bezerra et al. 2005, Brazilian Journal of Medical and BiologicalResearch 38: 161-170), including inflammatory arthritis (McClung 2006,Current Osteoporosis Reports 4: 28-33), Collagen-induced arthritis(Bezerra et al. 2005, Brazilian Journal of Medical and BiologicalResearch 38: 161-170); Periprosthetic osteolysis (McClung 2006, CurrentOsteoporosis Reports 4: 28-33; Anandarajah and Schwarz 2006, J. CellBiochem. 97: 226-232); Cancer-related bone disease (McClung 2006,Current Osteoporosis Reports 4: 28-33); Bone loss associated witharomatase inhibitor therapy (Lewiecki 2006, Expert Opin. Biol. Ther. 6:1041-1050); Bone loss associated with androgen deprivation therapy(Lewiecki 2006, Expert Opin. Biol. Ther. 6: 1041-1050); Bone lossassociated bone metastasis; Bone loss associated with diseases havingimmune system involvement, such as adult and childhood leukaemias,cancer metastasis, autoimmunity, and various viral infections (HolsteadJones et al. 2002, Ann. Rheum. Dis. 61 (Suppl II): ii32-ii39) Osteopenicdisorders such as adult and childhood leukaemia (Oliveri et al. 1999,Henry Ford Hosp. Med. 39: 45-48), chronic infections such as hepatitis Cor HIV (Stellon et al. 1985, Gastroenterology 89: 1078-1083), autoimmunedisorders such as diabetes mellitus (Piepkorn et al. 1997, Horm. Metab.Res. 29: 584-91), and lupus erythematosus (Seitz et al. 1985, Ann RheumDis. 44: 438-445), allergic diseases such as asthma (Ebeling et al.1998, J. Bone M M. Res. 13: 1283-1289), lytic bone metastases inmultiple cancers such as breast cancer (Coleman 1998, Curr. Opin. Oncol.10 (Suppl 1): 7-13); Prostate cancer; Myeloma bone disease (Anandarajahand Schwarz 2006, J. Cell Biochem. 97: 226-232); Periodontal infections(Anandarajah and Schwarz 2006, J. Cell Biochem. 97: 226-232); Expansileskeletal hyperphosphatasia (Anandarajah and Schwarz 2006, J. CellBiochem. 97: 226-232); Bone metastases (Lewiecki 2006, Expert Opin.Biol. Ther. 6: 1041-1050; Anandarajah and Schwarz 2006, J. Cell Biochem.97: 226-232).

Also encompassed within the scope of the present invention is theprevention and/or treatment with the amino acid sequences, the compoundsand/or the polypeptides of the invention of other diseases and disordersassociated with an imbalance in the RANK-L/RANK/OPG pathway. Suchdiseases and disorders include but are not limited to osteoporosis,inflammatory conditions, autoimmune conditions, asthma, rheumatoidarthritis, multiple sclerosis, Multiple myeloma (Sordillo and Pearse2003, Cancer 97 (3 Suppl): 802-812; Vanderkerken et al. 2003, CancerRes. 63: 287-289); Vascular diseases (Anandarajah and Schwarz 2006, J.Cell Biochem. 97: 226-232) and Cardiovascular disease (Lewiecki 2006,Expert Opin. Biol. Ther. 6: 1041-1050).

Also encompassed within the scope of the present invention is theprevention and/or treatment with the amino acid sequences, the compoundsand/or the polypeptides of the invention of diseases and disordersassociated with osteopetrosis such as osteopetrosis tarda, osteopetrosiscongenita and marble bone disease.

In particular, the polypeptides and compositions of the presentinvention can be used for the prevention and treatment of bone diseasesand disorders which are mediated by the pathway(s) in which RANK-L isinvolved. Examples of such bone diseases and disorders will again beclear to the skilled person based on the disclosure herein.

Thus, without being limited thereto, the amino acid sequences andpolypeptides of the invention can for example be used to prevent and/orto treat all diseases and disorders that are currently being preventedor treated with active principles that can modulate RANK-L-mediatedsignalling, such as those mentioned in the prior art cited above. It isalso envisaged that the polypeptides of the invention can be used toprevent and/or to treat all diseases and disorders for which treatmentwith such active principles is currently being developed, has beenproposed, or will be proposed or developed in future. In addition, it isenvisaged that, because of their favourable properties as furtherdescribed herein, the polypeptides of the present invention may be usedfor the prevention and treatment of other diseases and disorders thanthose for which these known active principles are being used or will beproposed or developed; and/or that the polypeptides of the presentinvention may provide new methods and regimens for treating the diseasesand disorders described herein.

Thus, without being limited thereto, the amino acid sequences andpolypeptides of the invention can for example be used to prevent and/orto treat all diseases and disorders that are currently being preventedor treated with Denosumab.

Other applications and uses of the amino acid sequences and polypeptidesof the invention will become clear to the skilled person from thefurther disclosure herein.

Generally, it is an object of the invention to provide pharmacologicallyactive agents, as well as compositions comprising the same, that can beused in the diagnosis, prevention and/or treatment of bone diseases anddisorders and of the further diseases and disorders mentioned herein;and to provide methods for the diagnosis, prevention and/or treatment ofsuch diseases and disorders that involve the administration and/or useof such agents and compositions.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions and/or methods that havecertain advantages compared to the agents, compositions and/or methodsthat are currently used and/or known in the art. These advantages willbecome clear from the further description below.

More in particular, it is an object of the invention to providetherapeutic proteins that can be used as pharmacologically activeagents, as well as compositions comprising the same, for the diagnosis,prevention and/or treatment of bone diseases and disorders and of thefurther diseases and disorders mentioned herein; and to provide methodsfor the diagnosis, prevention and/or treatment of such diseases anddisorders that involve the administration and/or the use of suchtherapeutic proteins and compositions.

Accordingly, it is a specific object of the present invention to provideamino acid sequences that are directed against (as defined herein)RANK-L, in particular against RANK-L from a warm-blooded animal, more inparticular against RANK-L from a mammal, and especially against humanRANK-L; and to provide proteins and polypeptides comprising oressentially consisting of at least one such amino acid sequence.

In particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat are suitable for prophylactic, therapeutic and/or diagnostic use ina warm-blooded animal, and in particular in a mammal, and more inparticular in a human being.

More in particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat can be used for the prevention, treatment, alleviation and/ordiagnosis of one or more diseases, disorders or conditions associatedwith RANK-L and/or mediated by RANK-L (such as the diseases, disordersand conditions mentioned herein) in a warm-blooded animal, in particularin a mammal, and more in particular in a human being.

It is also a specific object of the invention to provide such amino acidsequences and such proteins and/or polypeptides that can be used in thepreparation of pharmaceutical or veterinary compositions for theprevention and/or treatment of one or more diseases, disorders orconditions associated with and/or mediated by RANK-L (such as thediseases, disorders and conditions mentioned herein) in a warm-bloodedanimal, in particular in a mammal, and more in particular in a humanbeing.

In the invention, generally, these objects are achieved by the use ofthe amino acid sequences, proteins, polypeptides and compositions thatare described herein.

In general, the invention provides amino acid sequences that aredirected against (as defined herein) and/or can specifically bind (asdefined herein) to RANK-L; as well as compounds and constructs, and inparticular proteins and polypeptides, that comprise at least one suchamino acid sequence.

More in particular, the invention provides amino acid sequences can bindto RANK-L with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein; as well ascompounds and constructs, and in particular proteins and polypeptides,that comprise at least one such amino acid sequence.

In particular, amino acid sequences and polypeptides of the inventionare preferably such that they:

-   -   bind to RANK-L with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to RANK-L with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more        preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between        10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to RANK-L with a k_(off) rate between 1 s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶        s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or apolypeptide that contains only one amino acid sequence of the invention)is preferably such that it will bind to RANK-L with an affinity lessthan 500 nM, preferably less than 200 nM, more preferably less than 10nM, such as less than 500 pM.

Some preferred EC50 and IC50 values for binding of the amino acidsequences or polypeptides of the invention to RANK-L will become clearfrom the further description and examples herein.

For binding to RANK-L, an amino acid sequence of the invention willusually contain within its amino acid sequence one or more amino acidresidues or one or more stretches of amino acid residues (i.e. with each“stretch” comprising two or amino acid residues that are adjacent toeach other or in close proximity to each other, i.e. in the primary ortertiary structure of the amino acid sequence) via which the amino acidsequence of the invention can bind to RANK-L, which amino acid residuesor stretches of amino acid residues thus form the “site” for binding toRANK-L (also referred to herein as the “antigen binding site”).

The amino acid sequences provided by the invention are preferably inessentially isolated form (as defined herein), or form part of a proteinor polypeptide of the invention (as defined herein), which may compriseor essentially consist of one or more amino acid sequences of theinvention and which may optionally further comprise one or more furtheramino acid sequences (all optionally linked via one or more suitablelinkers). For example, and without limitation, the one or more aminoacid sequences of the invention may be used as a binding unit in such aprotein or polypeptide, which may optionally contain one or more furtheramino acid sequences that can serve as a binding unit (i.e. against oneor more other targets than RANK-L), so as to provide a monovalent,multivalent or multispecific polypeptide of the invention, respectively,all as described herein. Such a protein or polypeptide may also be inessentially isolated form (as defined herein).

The amino acid sequences and polypeptides of the invention as suchpreferably essentially consist of a single amino acid chain that is notlinked via disulphide bridges to any other amino acid sequence or chain(but that may or may not contain one or more intramolecular disulphidebridges. For example, it is known that NANOBODIES—as describedherein—may sometimes contain a disulphide bridge between CDR3 and CDR1or FR2). However, it should be noted that one or more amino acidsequences of the invention may be linked to each other and/or to otheramino acid sequences (e.g. via disulphide bridges) to provide peptideconstructs that may also be useful in the invention (for example Fab′fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies” and othermultispecific constructs. Reference is for example made to the review byHolliger and Hudson, Nat Biotechnol. 2005 September; 23(9):1126-36).

Generally, when an amino acid sequence of the invention (or a compound,construct or polypeptide comprising the same) is intended foradministration to a subject (for example for therapeutic and/ordiagnostic purposes as described herein), it is preferably either anamino acid sequence that does not occur naturally in said subject; or,when it does occur naturally in said subject, in essentially isolatedform (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use,the amino acid sequences of the invention (as well as compounds,constructs and polypeptides comprising the same) are preferably directedagainst human RANK-L; whereas for veterinary purposes, the amino acidsequences and polypeptides of the invention are preferably directedagainst RANK-L from the species to be treated, or at leastcross-reactive with RANK-L from the species to be treated.

Furthermore, an amino acid sequence of the invention may optionally, andin addition to the at least one binding site for binding against RANK-L,contain one or more further binding sites for binding against otherantigens, proteins or targets.

The efficacy of the amino acid sequences and polypeptides of theinvention, and of compositions comprising the same, can be tested usingany suitable in vitro assay, cell-based assay, in vivo assay and/oranimal model known per se, or any combination thereof, depending on thespecific disease or disorder involved. Suitable in vitro assays will beclear to the skilled person, and for example include ELISA; FACS bindingassay; BIACORE (surface plasmon resonance); competition binding assay(AlphaScreen®, Perkin Elmer, Massachusetts, USA; FMAT); TRAP assay(osteoclast differentiation assay; Rissanen et al. 2005, J. Bone Miner.Res. 20, Suppl. 1: S256); NF-kappaB reporter gene assay (Mizukami et al.2002, Mol. Cell. Biol. 22: 992-1000). EC50 values for binding of theNANOBODIES of the invention (and of polypeptides comprising the same) toRANK-L in, for example ELISA or FACS are preferably 1 μM to 1 pM, morepreferably 1 nM to 1 pM and more preferably 100 pM to 1 pM. IC50 valuesfor binding of the NANOBODIES of the invention (and of polypeptidescomprising the same) to RANK-L in, for example, AlphaScreen®, NF-kappaBassay or TRAP assay are preferably 1 μM to 1 pM, more preferably 1 nM to1 pM and more preferably 100 pM to 1 pM.

Suitable animal models will be clear to the skilled person, and forexample include (SCID)/ARH-77 mouse model (Sordillo and Pearse 2003,Cancer 97 (3 Suppl): 802-812); SCID-hu mouse model of human MM (Sordilloand Pearse 2003, Cancer 97 (3 Suppl): 802-812; Tassone et al. 2005,Blood 106: 713-716); Transgenic mice that overexpress OPG under controlof apoE gene promoter and associated enhancer (Simonet et al. 1997, Cell89: 309-319); Mouse model of sarcoma-induced bone destruction (Honore etal. 2000, Nat. Med. 6: 521-528); Ovariectomized animal models such as,for example, ovariectomized monkeys (Jerome et al. 1995, Bone 17:403S-408S), ovariectomized mice (Roggia et al. 2001, Proc. Natl. Acad.Sci. USA 20: 13960-13965) or ovariectomized rats and cynomolgus monkeys(Simonet et al. 1997, Cell 89: 309-319; Høegh-Andersen et al. 2004,Arthritis Res. Ther. 6: R169-R180); Rat (animal) models for arthritis(Bendele et al. 1999, Toxicologic Pathology 27: 134-142; Romas et al.2002, Am. J. Pathol. 161: 1419-1427; Mori et al. 2002, Histochemistryand Cell Biology 117: 283-292) such as models for collagen-inducedarthritis or models for adjuvant-induced arthritis; Animal models oftumor-derived PTHrP-induced hypercalcemia (Morony et al. 1999, J. BoneMiner. Res. 14: 1478-1485; Capparelli et al. 2000, Cancer Res. 60:783-778); Murine model of multiple myeloma (Vanderkerken et al. 2003,Cancer Res. 63: 287-289); Inflammatory Bowel Disease model in mice(Byrne et al. 2005, Gut 54: 78-86); Transgenic mice overexpressing MIF(Onodera et al. 2006, J. Bone Miner. Res. 21: 876-885); Transgenic miceoverexpressing soluble osteoclast differentiation factor (sODF) (Mizunoet al. 2002, 20: 337-44); Transgenic mice expressing CSF-1 under controlof the CSF-1R promoter/first intron driver [transgene TgN(Csf1r-Csf1)Ers(TgRC) mice] (Wei et al. 2006, J. Leukoc. Biol. 80: 1445-1453);Transgenic mice overexpressing core-binding factor alphal (Cbfal)(Geoffroy et al. Mol. Cell Biol. 22: 6222-6233); Transgenic miceoverexpressing Decoy receptor 3 (DcR3) (Tang et al. 2007, J. Biol. Chem.282: 2346-2354), as well as the assays and animal models used in theexperimental part below and in the prior art cited herein.

Also, according to the invention, amino acid sequences and polypeptidesthat are directed against RANK-L from a first species of warm-bloodedanimal may or may not show cross-reactivity with RANK-L from one or moreother species of warm-blooded animal. For example, amino acid sequencesand polypeptides directed against human RANK-L may or may not show crossreactivity with RANK-L from one or more other species of primates (suchas, without limitation, monkeys from the genus Macaca (such as, and inparticular, cynomolgus monkeys (Macaca fascicularis) and/or rhesusmonkeys (Macaca mulatta)) and baboon (Papio ursinus)) and/or with RANK-Lfrom one or more species of animals that are often used in animal modelsfor diseases (for example mouse, rat, rabbit, pig or dog), and inparticular in animal models for diseases and disorders associated withRANK-L (such as the species and animal models mentioned herein). In thisrespect, it will be clear to the skilled person that suchcross-reactivity, when present, may have advantages from a drugdevelopment point of view, since it allows the amino acid sequences andpolypeptides against human RANK-L to be tested in such disease models.

More generally, amino acid sequences and polypeptides of the inventionthat are cross-reactive with RANK-L from multiple species of mammal willusually be advantageous for use in veterinary applications, since itwill allow the same amino acid sequence or polypeptide to be used acrossmultiple species. Thus, it is also encompassed within the scope of theinvention that amino acid sequences and polypeptides directed againstRANK-L from one species of animal (such as amino acid sequences andpolypeptides against human RANK-L) can be used in the treatment ofanother species of animal, as long as the use of the amino acidsequences and/or polypeptides provide the desired effects in the speciesto be treated.

In one embodiment, the amino acid sequences and polypeptides of theinvention directed against human RANK-L are cross-reactive with RANK-Lfrom cynomolgus monkey.

In another embodiment, the amino acid sequences and polypeptides of theinvention directed against human RANK-L are cross-reactive with RANK-Lfrom mice or rats.

In another embodiment, the amino acid sequences and polypeptides of theinvention directed against human RANK-L are cross-reactive with RANK-Lfrom cynomolgus monkey and with RANK-L from mice or rats.

In another embodiment, the amino acid sequences and polypeptides of theinvention directed against human RANK-L are not cross-reactive withRANK-L from mice or rats.

In another embodiment, the amino acid sequences and polypeptides of theinvention directed against human RANK-L are cross-reactive with RANK-Lfrom cynomolgus monkey while not being cross-reactive with RANK-L frommice or rats.

In another embodiment, the amino acid sequences and polypeptides of theinvention directed against human RANK-L are not cross-reactive withRANK-L from cynomolgus monkey.

In another embodiment, the amino acid sequences and polypeptides of theinvention directed against human RANK-L are not cross-reactive withRANK-L from cynomolgus monkey and not with RANK-L from mice or rats.

The present invention is in its broadest sense also not particularlylimited to or defined by a specific antigenic determinant, epitope,part, domain, subunit or confirmation (where applicable) of RANK-Lagainst which the amino acid sequences and polypeptides of the inventionare directed. For example, the amino acid sequences and polypeptides mayor may not be directed against an “interaction site” (as definedherein). However, it is generally assumed and preferred that the aminoacid sequences and polypeptides of the invention are preferably directedagainst an interaction site (as defined herein), and in particularagainst the binding site on RANK-L for RANK or against the binding siteon RANK-L for OPG.

Thus, in one preferred, but non-limiting aspect, the amino acidsequences and polypeptides of the invention are directed against theRANK receptor binding site on RANK-L, and are as further defined herein.Binding of the amino acid sequences and polypeptides of the invention tothe RANK receptor binding site on RANK-L may inhibit and/or preventbinding of RANK-L to RANK, and thus inhibit or prevent the signallingthat is mediated by this RANK-L/RANK binding. The amino acid sequencesand polypeptides of the invention therefore can act as antagonists ofthe RANK-L/RANK mediated signalling.

In one, specific, but non-limiting aspect, the amino acid sequences andpolypeptides of the invention are directed against the RANK receptorbinding site on RANK-L while not interfering (reducing/inhibiting) withthe RANK-L/OPG interaction. In this specific aspect, the amino acidsequences and polypeptides of the invention will act as antagonists ofthe RANK-L/RANK mediated signalling (and inhibit osteoclast maturationand activation) in addition to the OPG mediated inhibition of osteoclastmaturation and activation.

In another preferred, but non-limiting aspect, the amino acid sequencesand polypeptides of the invention are directed against the OPG bindingsite on RANK-L, and are as further defined herein. Binding of the aminoacid sequences and polypeptides of the invention to the OPG binding siteon RANK-L may inhibit and/or prevent binding of RANK-L to OPG. The aminoacid sequences and polypeptides of the invention may therefore act as acompetitive or as a non-competitive inhibitor of the binding of RANK-Lto OPG (e.g. in ELISA, in AlphaScreen® assay, in TRAP assay and/or inNFkappaB assay). Binding of the amino acid sequences and polypeptides ofthe invention to the OPG binding site on RANK-L may, by its turn,inhibit and/or prevent binding of RANK-L to RANK, and thus inhibitand/or prevent the signalling that is mediated by this RANK-L/RANKbinding. The amino acid sequences and polypeptides of the inventiontherefore can act as antagonists of the RANK-L and RANK-L/RANK mediatedsignalling (i.e. they inhibit RANK/RANK-L interaction).

In some cases, however, the amino acid sequences and polypeptides of theinvention may be directed against an OPG binding site on RANK-L andinterfere with the binding of RANK-L to OPG without essentially reducingthe binding of RANK-L to RANK. In this case the amino acid sequences andpolypeptides of the invention may boost the signalling that is mediatedby this RANK-L/RANK interaction and act as agonists of the RANK-L and

RANK-L/RANK mediated signalling (i.e. they act as antagonist of theaction of OPG).

In another aspect of the present invention, the amino acid sequences andpolypeptides of the invention are preferably directed against an epitopeon RANK-L that overlaps with the epitope of Denosumab. Binding of theamino acid sequences and polypeptides of the invention to an epitope onRANK-L that overlaps with the epitope of Denosumab may inhibit and/orprevent binding of Denosumab to RANK-L. The amino acid sequences andpolypeptides of the invention may therefore act as a competitive or as anon-competitive inhibitor of the binding of Denosumab to RANK-L (e.g. inELISA, in AlphaScreen® assay, in TRAP assay and/or in NFkappaB assay).

As further described herein, a polypeptide of the invention may containtwo or more amino acid sequences of the invention that are directedagainst RANK-L. Generally, such polypeptides will bind to RANK-L withincreased avidity compared to a single amino acid sequence of theinvention. Such a polypeptide may for example comprise two amino acidsequences of the invention that are directed against the same antigenicdeterminant, epitope, part, domain, subunit or confirmation (whereapplicable) of RANK-L (which may or may not be an interaction site); orcomprise at least one “first” amino acid sequence of the invention thatis directed against a first same antigenic determinant, epitope, part,domain, subunit or confirmation (where applicable) of RANK-L (which mayor may not be an interaction site); and at least one “second” amino acidsequence of the invention that is directed against a second antigenicdeterminant, epitope, part, domain, subunit or confirmation (whereapplicable) different from the first (and which again may or may not bean interaction site). Preferably, in such “biparatopic” polypeptides ofthe invention, at least one amino acid sequence of the invention isdirected against an interaction site (as defined herein), although theinvention in its broadest sense is not limited thereto.

Thus, in one particular aspect, a polypeptide of the invention maycomprise two or more amino acid sequences of the invention that aredirected against the binding site for RANK on RANK-L; or comprise atleast one “first” amino acid sequence of the invention that is directedagainst the binding site for RANK on RANK-L; and at least one “second”amino acid sequence of the invention that is directed against a secondantigenic determinant, epitope, part, domain, subunit or confirmationdifferent from the first and which is not a binding site for RANK onRANK-L.

Thus, in another particular aspect, a polypeptide of the invention maycomprise two or more amino acid sequences of the invention that aredirected against the binding site for OPG on RANK-L; or comprise atleast one “first” amino acid sequence of the invention that is directedagainst the binding site for OPG on RANK-L; and at least one “second”amino acid sequence of the invention that is directed against a secondantigenic determinant, epitope, part, domain, subunit or confirmationdifferent from the first and which is not a binding site for OPG onRANK-L.

Also, when the target is part of a binding pair (for example, areceptor-ligand binding pair), the amino acid sequences and polypeptidesmay be such that they compete with the cognate binding partner (e.g. theligand, receptor or other binding partner, as applicable) for binding tothe target, and/or such that they (fully or partially) neutralizebinding of the binding partner to the target.

It is also within the scope of the invention that, where applicable, anamino acid sequence of the invention can bind to two or more antigenicdeterminants, epitopes, parts, domains, subunits or confirmations ofRANK-L. In such a case, the antigenic determinants, epitopes, parts,domains or subunits of RANK-L to which the amino acid sequences and/orpolypeptides of the invention bind may be essentially the same (forexample, if RANK-L contains repeated structural motifs or occurs in amultimeric form) or may be different (and in the latter case, the aminoacid sequences and polypeptides of the invention may bind to suchdifferent antigenic determinants, epitopes, parts, domains, subunits ofRANK-L with an affinity and/or specificity which may be the same ordifferent). In a preferred, but non-limiting aspect, the amino acidsequences and polypeptides of the invention bind two or three subunitsof the RANK-L trimer. Also, for example, the amino acid sequences andpolypeptides of the invention may bind to a conformation of RANK-L inwhich it is bound to a pertinent ligand, may bind to a conformation ofRANK-L in which it not bound to a pertinent ligand, or may bind to bothsuch conformations (again with an affinity and/or specificity which maybe the same or different). For example, the amino acid sequences andpolypeptides of the invention may bind to a conformation of RANK-L inwhich it is bound to RANK, may bind to a conformation of RANK-L in whichit not bound to RANK, or may bind to both such conformations (again withan affinity and/or specificity which may be the same or different). Forexample, the amino acid sequences and polypeptides of the invention maybind to a conformation of RANK-L in which it is bound to OPG, may bindto a conformation of RANK-L in which it not bound to OPG, or may bind toboth such conformations (again with an affinity and/or specificity whichmay be the same or different).

RANK-L exists in a membrane bound and soluble form. The amino acidsequences and polypeptides of the invention may bind to either forms, orpreferably the amino acid sequences and polypeptides of the inventionmay bind to both these forms. RANK-L exists in four different isoforms(see supra). The amino acid sequences and polypeptides of the inventionmay bind to either one of the four isoforms of RANK-L, or may bind tomore than one such as two, three or all four isoforms of RANK-L.

It is also expected that the amino acid sequences and polypeptides ofthe invention will generally bind to all naturally occurring orsynthetic analogs, variants, mutants, alleles, parts and fragments ofRANK-L; or at least to those analogs, variants, mutants, alleles, partsand fragments of RANK-L that contain one or more antigenic determinantsor epitopes that are essentially the same as the antigenicdeterminant(s) or epitope(s) to which the amino acid sequences andpolypeptides of the invention bind in RANK-L (e.g. in wild-type RANK-L).Again, in such a case, the amino acid sequences and polypeptides of theinvention may bind to such analogs, variants, mutants, alleles, partsand fragments with an affinity and/or specificity that are the same as,or that are different from (i.e. higher than or lower than), theaffinity and specificity with which the amino acid sequences of theinvention bind to (wild-type) RANK-L. It is also included within thescope of the invention that the amino acid sequences and polypeptides ofthe invention bind to some analogs, variants, mutants, alleles, partsand fragments of RANK-L, but not to others.

The amino acid sequences and polypeptides of the invention may bind toother, related TNF family members (e.g. TRAIL, TNF-alpha and/or CD40ligand). In a preferred aspect, however, the amino acid sequences andpolypeptides of the invention will have no detectable affinity forrelated TNF family members (i.e. an affinity which is more than 10times, preferably more than 100 times, more preferably more than 1000times lower than its affinity for RANK-L). In one aspect, the amino acidsequences and polypeptides of the invention will have no detectableaffinity for TRAIL. In another aspect, the amino acid sequences andpolypeptides of the invention will have no detectable affinity forTNF-alpha. In another aspect, the amino acid sequences and polypeptidesof the invention will have no detectable affinity for CD40 ligand. Inyet another aspect, the amino acid sequences and polypeptides of theinvention will have no detectable affinity for TRAIL, TNF-alpha and/orCD40 ligand.

Similar to all known TNF cytokine family members, RANK-L self-assemblesinto noncovalently associated trimers. When RANK-L exists in a monomericform and in one or more multimeric forms, it is within the scope of theinvention that the amino acid sequences and polypeptides of theinvention only bind to RANK-L in monomeric form, only bind to RANK-L inmultimeric form, or bind to both the monomeric and the multimeric form.Again, in such a case, the amino acid sequences and polypeptides of theinvention may bind to the monomeric form with an affinity and/orspecificity that are the same as, or that are different from (i.e.higher than or lower than), the affinity and specificity with which theamino acid sequences of the invention bind to the multimeric form.

In one non-limiting aspect of the invention, the amino acid sequencesand polypeptides of the invention bind to RANK-L such that the formationof the RANK-L trimer is prevented and/or inhibited.

It is accepted that RANK-L binds one receptor molecule (RANK) along eachof the three clefts (or grooves) formed by neighboring monomers of thehomotrimer. In this way, the RANK-L trimer exhibits three spatiallydistinct, but equivalent, intersubunit receptor-binding grooves intowhich three receptor molecules bind. Therefore, in order to inhibit theinteraction between RANK-L and its receptors, therapeutic moleculesshould preferably target these intersubunit receptor binding grooves ofRANK-L. NANOBODIES (as further defined herein) can show so-calledcavity-binding properties (inter alia due to their extended CDR3 loop,compared to conventional V_(H) domains) and are therefore ideally suitedfor inhibition of the interaction of RANK-L with its RANK receptor.Accordingly, in a preferred aspect, the amino acid sequences andpolypeptides of the invention bind to the intersubunit receptor-bindinggrooves of RANK-L

Also, when RANK-L can associate with other proteins or polypeptides toform protein complexes (e.g. with multiple subunits), it is within thescope of the invention that the amino acid sequences and polypeptides ofthe invention bind to RANK-L in its non-associated state, bind to RANK-Lin its associated state, or bind to both. In all these cases, the aminoacid sequences and polypeptides of the invention may bind to suchmultimers or associated protein complexes with an affinity and/orspecificity that may be the same as or different from (i.e. higher thanor lower than) the affinity and/or specificity with which the amino acidsequences and polypeptides of the invention bind to RANK-L in itsmonomeric and non-associated state.

Generally, amino acid sequences and polypeptides of the invention willat least bind to those forms of RANK-L (including monomeric, multimericand associated forms) that are the most relevant from a biologicaland/or therapeutic point of view, as will be clear to the skilledperson.

Also, generally, polypeptides of the invention that contain two or moreamino acid sequences and/or NANOBODIES directed against RANK-L may bindwith higher avidity than the corresponding monomeric amino acidsequences and/or NANOBODIES.

For example, and without limitation, a multivalent (as defined herein)protein or polypeptide that contains two or more amino acid sequencesand/or NANOBODIES that are directed against different epitopes of RANK-Lmay bind to RANK-L with higher avidity than the corresponding monomers.

More importantly, a multivalent (as defined herein) protein orpolypeptide that contains two or more amino acid sequences and/orNANOBODIES that are directed against RANK-L may (and usually will) bindwith higher avidity to a multimer of RANK-L than to a monomer of RANK-L,and will usually also bind with higher avidity than the correspondingmonomeric amino acid sequences and/or NANOBODIES. In such a multivalentprotein or polypeptide, the two or more amino acid sequences and/orNANOBODIES may for example be directed against the same epitopes,substantially equivalent epitopes, or different epitopes. In oneembodiment of such a multivalent protein or polypeptide, the two or moreamino acid sequences and/or NANOBODIES may be the same (and therefore bedirected against the same epitope).

The latter is of particular importance, as it is known that the primarymode of signal transduction by RANK-L involves binding of RANK receptorsto a trimer of RANK-L molecules, which contains three receptor bindingsites (see for example Lam et al. 2001, J. Clin. Invest. 108: 971-979).

In the present invention, it has been found that amino acid sequencesand/or NANOBODIES are capable of binding to RANK-L in such a way thatthe activity of RANK-L is reduced, both in in vitro models and incellular models (see the Experimental Section below). Although theinvention is not limited to any specific mechanism, explanation orhypothesis, it is assumed that because of their small size and highaffinity for RANK-L, two or three monovalent amino acid sequences and/orNANOBODIES of the invention are capable of simultaneously occupying twoor three different receptor binding sites on the RANK-L trimer, thuspreventing the trimer to initiate receptor binding and thereby toinitiate signal transduction (however, other mechanisms of action arenot excluded: for example, depending on the epitope against which it isdirected, an amino acid sequence and/or NANOBODY of the invention mayalso inhibit the association of RANK-L into the trimeric state).

It should also be noted that, in addition or as an alternative tobinding to two or more receptor binding sites on a single RANK-L trimer,the proteins or polypeptides of the present invention that comprises oressentially consists of two or more amino acid sequences and/orNANOBODIES that are directed against epitopes of RANK-L may bind (e.g.intermolecularly) epitopes on two separate RANK-L molecules (e.g. twoseparate trimers).

However, according to one particularly preferred embodiment, theinvention relates to a protein or polypeptide that comprises oressentially consists of two or more amino acid sequences and/orNANOBODIES that are each directed against epitopes on RANK-L (and inparticular on the RANK-L trimer) that lie in and/or form part of thereceptor binding site(s) of the RANK-L trimer, such that saidpolypeptide, upon binding to a RANK-L trimer, is capable of inhibitingor reducing the RANK receptor binding that is mediated by said RANK-Ltrimer and/or the signal transduction that is mediated by such receptorbinding.

In particular, according to this preferred embodiment, the inventionrelates to a protein or polypeptide that comprises or essentiallyconsist of two or more amino acid sequences and/or NANOBODIES that areeach directed against epitopes on RANK-L (and in particular on theRANK-L trimer) that lie in and/or form part of the receptor bindingsite(s) of the RANK-L trimer, wherein said amino acid sequences and/orNANOBODIES are linked to each other in such a way that the protein orpolypeptide is capable of simultaneously binding to two or more receptorbinding sites on a single RANK-L trimer (in other words, is capable ofintramolecular binding to at least two RANK-L receptor binding sites ona RANK-L trimer). In this embodiment, the two or more amino acidsequences and/or NANOBODIES are preferably as defined above and are mostpreferably NANOBODIES (so that the protein or polypeptide is amultivalent NANOBODY construct, as further described herein). Also, inthis embodiment, the two or more amino acid sequences and/or NANOBODIESmay be the same or different; and may directed against differentepitopes within the RANK receptor binding site(s), but are preferablydirected against the same epitope. Some preferred, but non-limitingconstructs of this embodiment of the invention are SEQ ID NO's: 622 to693, 761 to 762 and 766 to 773.

In this embodiment of the invention, the two or more amino acidsequences and/or NANOBODIES will usually be linked via one or moresuitable linkers, which linkers are such that each amino acid sequencesand/or NANOBODIES can bind to a different receptor binding site on thesame RANK-L trimer. Suitable linkers will inter alia depend on (thedistance between) the epitopes on the RANK-L trimer to which the aminoacid sequences and/or NANOBODIES bind, and will be clear to the skilledperson based on the disclosure herein, optionally after some limiteddegree of routine experimentation. For example, when the two or moreamino acid sequences are (single) domain antibodies or NANOBODIES,suitable linkers may be chosen from the linkers described herein, butwith a linker length that is such that the two or more (single) domainantibodies or NANOBODIES can each bind to a different receptor bindingsite on the same RANK-L trimer.

Also, when the two or more amino acid sequences that bind to thereceptor binding sites of RANK-L are (single) domain antibodies orNANOBODIES, they may also be linked to each other via a third (single)domain antibody or NANOBODY (in which the two or more (single) domainantibodies or NANOBODIES may be linked directly to the third (single)domain antibody/NANOBODY or via suitable linkers). Such a third (single)domain antibody or NANOBODY may for example be a (single) domainantibody or NANOBODY that provides for an increased half-life, asfurther described herein. For example, the latter (single) domainantibody or NANOBODY may be a (single) domain antibody or NANOBODY thatis capable of binding to a (human) serum protein such as (human) serumalbumin, as further described herein. Some non-limiting examples of suchconstructs are the constructs of SEQ ID NO's: 694-729 and 759-760. Sucha third (single) domain antibody or NANOBODY may for example be a(single) domain antibody or NANOBODY that is directed against and/or canbind another epitope on RANK-L, providing a biparatopic (single) domainantibody or NANOBODIES, as is further described herein.

Alternatively, the two or more amino acid sequences and/or NANOBODIESthat bind to the receptor binding site(s) of RANK-L may be linked inseries (either directly or via a suitable linker) and the third (single)domain antibody or NANOBODY (which may provide for increased half-lifeor which may bind another epitope on RANK-L, as described above) may beconnected directly or via a linker to one of these two or moreaforementioned amino acid sequences and/or NANOBODIES.

More generally, the distance between the two or more amino acidsequences and/or NANOBODIES should be such that it allows the protein orpolypeptide to undergo intramolecular binding to the RANK-L trimer (i.e.instead of intermolecular binding). The distance between the N-terminusand the C-terminus of two anti-RANK-L amino acid sequences and/orNANOBODIES can be determined by any suitable means, such as bycrystallography or molecular modelling (as described, for example, byLam et al. 2001, J. Clin. Invest. 108: 971-979). These techniquesgenerally also make it possible to determine whether a specificmultivalent or multispecific protein or polypeptide is capable ofproviding intramolecular modelling. Alternatively, size-exclusionchromatography (as described by Santora et al., Anal. Biochem., 299:119-129) could be used to determine whether a given protein orpolypeptide of the invention will (predominantly) provide intramolecularbinding to a RANK-L trimer or (predominantly) intermolecular bindingbetween two or more RANK-L trimers. Thus, in one particular embodimentof the invention, a protein or polypeptide of the invention ispreferably such that, in this experiment, it predominantly oressentially exclusively leads to intramolecular binding. However, asemphasized above, it should be noted that proteins or polypeptides ofthe invention that operate via intermolecular binding of separate RANK-Lmolecules (e.g. trimers) are also within the scope of the presentinvention.

Thus, in another preferred aspect, the invention provides for amultivalent or multispecific protein or polypeptide that comprises atleast two amino acid sequences and/or NANOBODIES against RANK-L (and inparticular of the RANK-L trimer), in which said at least two amino acidsequences and/or NANOBODIES are linked in such a way that the distancebetween the N-terminus and the C-terminus of the at least twoanti-RANK-L amino acid sequences and/or NANOBODIES is such that theprotein or polypeptide is capable of undergoing intramolecular binding(as described herein) with a RANK-L trimer.

In such a preferred protein or polypeptide, the two or more amino acidsequences and/or NANOBODIES may be linked in any suitable fashion, aslong as the preferred distance between the N-terminus and the C-terminusof the at least two anti-RANK-L amino acid sequences and/or NANOBODIEScan be achieved, and/or as long as the protein or polypeptide is capableof undergoing intramolecular binding (as described herein) with a RANK-Ltrimer.

For example, in its simplest form, the at least two amino acid sequencesand/or NANOBODIES are directly linked via a suitable linker or spacerthat provides for the preferred distance between the N-terminus and theC-terminus of the at least two anti-RANK-L amino acid sequences and/orNANOBODIES and which may allow the protein or polypeptide to undergointramolecular binding (as described herein) with a RANK-L trimer.Suitable linkers are described herein, and may—for example and withoutlimitation—comprise an amino acid sequence, which amino acid sequencepreferably has a length of from 1 up to 50 or more amino acids, morepreferably from 5 to 30 amino acids, such as about 9 to 20 amino acids.Preferably, such an amino acid sequence should also be such that itallows the protein or polypeptide to undergo intramolecular binding (asdescribed herein) with a RANK-L trimer.

Thus, in another preferred aspect, the invention provides for amultivalent or multispecific protein or polypeptide that comprises atleast two amino acid sequences and/or NANOBODIES against RANK-L (and inparticular the RANK-L trimer), in which said amino acid sequences and/orNANOBODIES are preferably directly linked to each other using a suitablelinker or spacer such that the distance between the N-terminus and theC-terminus of the at least two anti-RANK-L amino acid sequences and/orNANOBODIES is such that the protein or polypeptide is capable ofundergoing intramolecular binding (as described herein) with a RANK-Ltrimer.

More preferably, in this preferred aspect, the linker or spacer is anamino acid sequence that preferably has a length of from 1 up to 50 ormore amino acids, more preferably from 5 to 30 amino acids, such asabout 9 to 20 amino acids. In one preferred, but non-limitingembodiment, the linker essentially consists of glycine and serineresidues (as further described below). For example, one suitable linkeris the GS9 linker described herein, which comprises 9 amino acidresidues, the GS15 linker described herein, which comprises 15 aminoacid residues, the GS20 linker described herein, which comprises 20amino acid residues and the GS30 linker described herein, whichcomprises 30 amino acid residues.

In another embodiment, the at least two amino acid sequences and/orNANOBODIES against RANK-L are linked to each other via another moiety(optionally via one or two linkers), such as another protein orpolypeptide. In this embodiment, it may be desirable to have thepreferred distance (i.e. as mentioned above) between the N-terminus andthe C-terminus of the at least two anti-RANK-L amino acid sequencesand/or NANOBODIES, for example such that the protein or polypeptide canstill undergo intramolecular binding (as described herein) with a RANK-Ltrimer. In this embodiment, the at least two amino acid sequences and/orNANOBODIES may be linked directly to the other moiety, or using asuitable linker or spacer, again as long as the preferred distanceand/or desired intramolecular binding can still be achieved. The moietymay be any suitable moiety which does not detract (too much) from thebinding of the protein or polypeptide to RANK-L and/or from the furtherdesired biological or pharmacological properties of the protein orpolypeptide. As such, the moiety may be essentially inactive or may bebiologically active, and as such may or may not improve the desiredproperties of the protein or polypeptide and/or may confer one or moreadditional desired properties to the protein or polypeptide. Forexample, and without limitation, the moiety may improve the half-life ofthe protein or polypeptide, and/or may reduce its immunogenicity orimprove any other desired property. In one preferred embodiment, themoiety may be another amino acid sequences and/or NANOBODY (includingbut not limited to a third amino acid sequences and/or NANOBODY againstRANK-L, although this is not necessary and usually less preferred), andin particular another amino acid sequences and/or NANOBODY that improvesthe half-life of the protein or polypeptide, such as an amino acidsequences and/or NANOBODY that is directed against a serum protein, forexample against human serum albumin Examples of such proteins andpolypeptides are described herein.

Thus, in one embodiment, the invention relates to a multivalentmultispecific construct comprising two or more amino acid sequencesand/or NANOBODIES that are each directed against epitopes on RANK-L(e.g. on the RANK-L trimer) that lie in and/or form part of the receptorbinding site, and that are linked to each other via at least one aminoacid sequences and/or NANOBODY that provides for increased half-life(and optionally via one or more suitable linkers), such that saidpolypeptide, upon binding to a RANK-L trimer, is capable inhibiting orreducing the RANK receptor binding and/or the signal transduction thatis mediated by said RANK-L trimer. Such a polypeptide may be such thatsaid firstmentioned two or more amino acid sequences and/or NANOBODIEScan each bind to a different receptor binding site on a RANK-L trimer.

In particular, in this embodiment, the polypeptide may comprise atrivalent bispecific NANOBODY, that comprises two NANOBODIES that areeach directed against epitopes on RANK-L (and in particular of theRANK-L trimer) that lie in and/or form part of the receptor bindingsite, in which said NANOBODIES are linked to each other via a thirdNANOBODY that provides for an increased half-life (e.g. a NANOBODY thatis directed to a serum protein such as human serum albumin), in whicheach of the firstmentioned two NANOBODIES may be directly linked to saidthird NANOBODY or via one or more suitable linkers, such that saidpolypeptide, upon binding to a RANK-L trimer, is capable of inhibitingor reducing the RANK receptor binding and/or the signal transductionthat is mediated by said RANK-L trimer. Such a polypeptide may be suchthat said firstmentioned two NANOBODIES can each bind to a differentreceptor binding site on a RANK-L trimer. Again, some particularlypreferred NANOBODIES for use in this embodiment of the invention arepresented in SEQ ID NO's: 560 to 621, as well as humanized and othervariants thereof (such as e.g. SEQ ID NO's: 730 to 757 and 765); and theNANOBODIES directed against human serum albumin described herein. Somepreferred, but non-limiting constructs of this embodiment of theinvention are SEQ ID NO's: 694 to 729 and 759 to 760.

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of the amino acidsequences and polypeptides of the invention, and/or to use proteins orpolypeptides comprising or essentially consisting of one or more of suchparts, fragments, analogs, mutants, variants, alleles and/orderivatives, as long as these are suitable for the uses envisagedherein. Such parts, fragments, analogs, mutants, variants, allelesand/or derivatives will usually contain (at least part of) a functionalantigen-binding site for binding against RANK-L; and more preferablywill be capable of specific binding to RANK-L, and even more preferablycapable of binding to RANK-L with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined herein.Some non-limiting examples of such parts, fragments, analogs, mutants,variants, alleles, derivatives, proteins and/or polypeptides will becomeclear from the further description herein. Additional fragments orpolypeptides of the invention may also be provided by suitably combining(i.e. by linking or genetic fusion) one or more (smaller) parts orfragments as described herein.

In one specific, but non-limiting aspect of the invention, which will befurther described herein, such analogs, mutants, variants, alleles,derivatives have an increased half-life in serum (as further describedherein) compared to the amino acid sequence from which they have beenderived. For example, an amino acid sequence of the invention may belinked (chemically or otherwise) to one or more groups or moieties thatextend the half-life (such as PEG), so as to provide a derivative of anamino acid sequence of the invention with increased half-life.

In one specific, but non-limiting aspect, the amino acid sequence of theinvention may be an amino acid sequence that comprises an immunoglobulinfold or may be an amino acid sequence that, under suitable conditions(such as physiological conditions) is capable of forming animmunoglobulin fold (i.e. by folding). Reference is inter alia made tothe review by Halaby et al., J. (1999) Protein Eng. 12, 563-71.Preferably, when properly folded so as to form an immunoglobulin fold,such an amino acid sequence is capable of specific binding (as definedherein) to RANK-L; and more preferably capable of binding to RANK-L withan affinity (suitably measured and/or expressed as a K_(D)-value (actualor apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein. Also, parts, fragments, analogs,mutants, variants, alleles and/or derivatives of such amino acidsequences are preferably such that they comprise an immunoglobulin foldor are capable for forming, under suitable conditions, an immunoglobulinfold.

In particular, but without limitation, the amino acid sequences of theinvention may be amino acid sequences that essentially consist of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively); or any suitablefragment of such an amino acid sequence (which will then usually containat least some of the amino acid residues that form at least one of theCDR's, as further described herein).

The amino acid sequences of the invention may in particular be animmunoglobulin sequence or a suitable fragment thereof, and more inparticular be an immunoglobulin variable domain sequence or a suitablefragment thereof, such as light chain variable domain sequence (e.g. aV_(L)-sequence) or a suitable fragment thereof; or a heavy chainvariable domain sequence (e.g. a V_(H)-sequence) or a suitable fragmentthereof. When the amino acid sequence of the invention is a heavy chainvariable domain sequence, it may be a heavy chain variable domainsequence that is derived from a conventional four-chain antibody (suchas, without limitation, a V_(H) sequence that is derived from a humanantibody) or be a so-called V_(HH)-sequence (as defined herein) that isderived from a so-called “heavy chain antibody” (as defined herein).

However, it should be noted that the invention is not limited as to theorigin of the amino acid sequence of the invention (or of the nucleotidesequence of the invention used to express it), nor as to the way thatthe amino acid sequence or nucleotide sequence of the invention is (orhas been) generated or obtained. Thus, the amino acid sequences of theinvention may be naturally occurring amino acid sequences (from anysuitable species) or synthetic or semi-synthetic amino acid sequences.In a specific but non-limiting aspect of the invention, the amino acidsequence is a naturally occurring immunoglobulin sequence (from anysuitable species) or a synthetic or semi-synthetic immunoglobulinsequence, including but not limited to “humanized” (as defined herein)immunoglobulin sequences (such as partially or fully humanized mouse orrabbit immunoglobulin sequences, and in particular partially or fullyhumanized V_(HH) sequences or NANOBODIES), “camelized” (as definedherein) immunoglobulin sequences, as well as immunoglobulin sequencesthat have been obtained by techniques such as affinity maturation (forexample, starting from synthetic, random or naturally occurringimmunoglobulin sequences), CDR grafting, veneering, combining fragmentsderived from different immunoglobulin sequences, PCR assembly usingoverlapping primers, and similar techniques for engineeringimmunoglobulin sequences well known to the skilled person; or anysuitable combination of any of the foregoing. Reference is for examplemade to the standard handbooks, as well as to the further descriptionand prior art mentioned herein.

Similarly, the nucleotide sequences of the invention may be naturallyoccurring nucleotide sequences or synthetic or semi-synthetic sequences,and may for example be sequences that are isolated by PCR from asuitable naturally occurring template (e.g. DNA or RNA isolated from acell), nucleotide sequences that have been isolated from a library (andin particular, an expression library), nucleotide sequences that havebeen prepared by introducing mutations into a naturally occurringnucleotide sequence (using any suitable technique known per se, such asmismatch PCR), nucleotide sequence that have been prepared by PCR usingoverlapping primers, or nucleotide sequences that have been preparedusing techniques for DNA synthesis known per se.

The amino acid sequence of the invention may in particular be a domainantibody (or an amino acid sequence that is suitable for use as a domainantibody), a single domain antibody (or an amino acid sequence that issuitable for use as a single domain antibody), a “dAb” (or an amino acidsequence that is suitable for use as a dAb) or a NANOBODY (as definedherein, and including but not limited to a V_(HH) sequence); othersingle variable domains, or any suitable fragment of any one thereof.For a general description of (single) domain antibodies, reference isalso made to the prior art cited above, as well as to EP 0 368 684. Forthe term “dAb's”, reference is for example made to Ward et al. (Nature1989 Oct. 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol.,2003, 21(11):484-490; as well as to for example WO 06/030220, WO06/003388 and other published patent applications of Domantis Ltd. Itshould also be noted that, although less preferred in the context of thepresent invention because they are not of mammalian origin, singledomain antibodies or single variable domains can be derived from certainspecies of shark (for example, the so-called “IgNAR domains”, see forexample WO 05/18629).

In particular, the amino acid sequence of the invention may be aNANOBODY® (as defined herein) or a suitable fragment thereof. [Note:NANOBODY®, NANOBODIES® and NANOCLONE® are registered trademarks ofAblynx N.V.] Such NANOBODIES directed against RANK-L will also bereferred to herein as “NANOBODIES of the invention”.

For a general description of NANOBODIES, reference is made to thefurther description below, as well as to the prior art cited herein. Inthis respect, it should however be noted that this description and theprior art mainly described NANOBODIES of the so-called “V_(H)3 class”(i.e. NANOBODIES with a high degree of sequence homology to humangermline sequences of the V_(H)3 class such as DP-47, DP-51 or DP-29),which NANOBODIES form a preferred aspect of this invention. It shouldhowever be noted that the invention in its broadest sense generallycovers any type of NANOBODY directed against RANK-L, and for examplealso covers the NANOBODIES belonging to the so-called “V_(H)4 class”(i.e. NANOBODIES with a high degree of sequence homology to humangermline sequences of the V_(H)4 class such as DP-78), as for exampledescribed in WO 07/118670.

Generally, NANOBODIES (in particular V_(HH) sequences and partiallyhumanized NANOBODIES) can in particular be characterized by the presenceof one or more “Hallmark residues” (as described herein) in one or moreof the framework sequences (again as further described herein).

Thus, generally, a NANOBODY can be defined as an amino acid sequencewith the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which one or more of the Hallmark residues are as further        defined herein.

In particular, a NANOBODY can be an amino acid sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which the framework sequences are as further defined herein.

More in particular, a NANOBODY can be an amino acid sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below;    and in which:

-   ii) said amino acid sequence has at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 1 to    22, in which for the purposes of determining the degree of amino    acid identity, the amino acid residues that form the CDR sequences    (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are    disregarded.

In these NANOBODIES, the CDR sequences are generally as further definedherein.

Thus, the invention also relates to such NANOBODIES that can bind to (asdefined herein) and/or are directed against RANK-L, to suitablefragments thereof, as well as to polypeptides that comprise oressentially consist of one or more of such NANOBODIES and/or suitablefragments.

SEQ ID NO's: 560-621 give the amino acid sequences of a number of V_(HH)sequences that have been raised against RANK-L.

In particular, the invention in some specific aspects provides:

-   -   amino acid sequences that are directed against (as defined        herein) RANK-L and that have at least 80%, preferably at least        85%, such as 90% or 95% or more sequence identity with at least        one of the amino acid sequences of SEQ ID NO's: 560-621. These        amino acid sequences may further be such that they neutralize        binding of RANK or OPG to RANK-L; and/or compete with RANK or        OPG for binding to RANK-L; and/or are directed against an        interaction site (as defined herein) on RANK-L (such as the RANK        or OPG binding site);    -   amino acid sequences that cross-block (as defined herein) the        binding of at least one of the amino acid sequences of SEQ ID        NO's: 560-621 to RANK-L and/or that compete with at least one of        the amino acid sequences of SEQ ID NO's: 560-621 for binding to        RANK-L. Again, these amino acid sequences may further be such        that they neutralize binding of the cognate ligand to RANK-L;        and/or compete with the cognate ligand for binding to RANK-L;        and/or are directed against an interaction site (as defined        herein) on RANK-L (such as the RANK or OPG binding site);        which amino acid sequences may be as further described herein        (and may for example be NANOBODIES); as well as polypeptides of        the invention that comprise one or more of such amino acid        sequences (which may be as further described herein, and may for        example be bispecific and/or biparatopic polypeptides as        described herein), and nucleic acid sequences that encode such        amino acid sequences and polypeptides. Such amino acid sequences        and polypeptides do not include any naturally occurring ligands.

Accordingly, some particularly preferred NANOBODIES of the invention areNANOBODIES which can bind (as further defined herein) to and/or aredirected against to RANK-L and which:

-   i) have at least 80% amino acid identity with at least one of the    amino acid sequences of SEQ ID NO's: 560-621, in which for the    purposes of determining the degree of amino acid identity, the amino    acid residues that form the CDR sequences are disregarded. In this    respect, reference is also made to Table A-1, which lists the    framework 1 sequences (SEQ ID NO's: 126-187), framework 2 sequences    (SEQ ID NO's: 250-311), framework 3 sequences (SEQ ID NO's: 374-435)    and framework 4 sequences (SEQ ID NO's: 498-559) of the NANOBODIES    of SEQ ID NO's: 560-621 (with respect to the amino acid residues at    positions 1 to 4 and 27 to 30 of the framework 1 sequences,    reference is also made to the comments made below. Thus, for    determining the degree of amino acid identity, these residues are    preferably disregarded);    and in which:-   ii) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below.

In these NANOBODIES, the CDR sequences are generally as further definedherein.

Again, such NANOBODIES may be derived in any suitable manner and fromany suitable source, and may for example be naturally occurring V_(HH)sequences (i.e. from a suitable species of Camelid) or synthetic orsemi-synthetic amino acid sequences, including but not limited to“humanized” (as defined herein) NANOBODIES, “camelized” (as definedherein) immunoglobulin sequences (and in particular camelized heavychain variable domain sequences), as well as NANOBODIES that have beenobtained by techniques such as affinity maturation (for example,starting from synthetic, random or naturally occurring immunoglobulinsequences), CDR grafting, veneering, combining fragments derived fromdifferent immunoglobulin sequences, PCR assembly using overlappingprimers, and similar techniques for engineering immunoglobulin sequenceswell known to the skilled person; or any suitable combination of any ofthe foregoing as further described herein. Also, when a NANOBODYcomprises a V_(HH) sequence, said NANOBODY may be suitably humanized, asfurther described herein, so as to provide one or more further(partially or fully) humanized NANOBODIES of the invention. Similarly,when a NANOBODY comprises a synthetic or semi-synthetic sequence (suchas a partially humanized sequence), said NANOBODY may optionally befurther suitably humanized, again as described herein, again so as toprovide one or more further (partially or fully) humanized NANOBODIES ofthe invention.

In particular, humanized NANOBODIES may be amino acid sequences that areas generally defined for NANOBODIES in the previous paragraphs, but inwhich at least one amino acid residue is present (and in particular, inat least one of the framework residues) that is and/or that correspondsto a humanizing substitution (as defined herein). Some preferred, butnon-limiting humanizing substitutions (and suitable combinationsthereof) will become clear to the skilled person based on the disclosureherein. In addition, or alternatively, other potentially usefulhumanizing substitutions can be ascertained by comparing the sequence ofthe framework regions of a naturally occurring V_(HH) sequence with thecorresponding framework sequence of one or more closely related humanV_(H) sequences, after which one or more of the potentially usefulhumanizing substitutions (or combinations thereof) thus determined canbe introduced into said V_(HH) sequence (in any manner known per se, asfurther described herein) and the resulting humanized V_(HH) sequencescan be tested for affinity for the target, for stability, for ease andlevel of expression, and/or for other desired properties. In this way,by means of a limited degree of trial and error, other suitablehumanizing substitutions (or suitable combinations thereof) can bedetermined by the skilled person based on the disclosure herein. Also,based on the foregoing, (the framework regions of) a NANOBODY may bepartially humanized or fully humanized.

Some particularly preferred humanized NANOBODIES of the invention arehumanized variants of the NANOBODIES of SEQ ID NO's: 560-621, of whichthe amino acid sequences of SEQ ID NO's: 730-757 and 765 are someespecially preferred examples.

Thus, some other preferred NANOBODIES of the invention are NANOBODIESwhich can bind (as further defined herein) to RANK-L and which:

-   i) are a humanized variant of one of the amino acid sequences of SEQ    ID NO's: 560-621; and/or-   ii) have at least 80% amino acid identity with at least one of the    amino acid sequences of SEQ ID NO's: 560-621 and/or at least one of    the amino acid sequences of SEQ ID NO's: 730-757 and 765, in which    for the purposes of determining the degree of amino acid identity,    the amino acid residues that form the CDR sequences are disregarded;    and in which:-   i) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below.

According to another specific aspect of the invention, the inventionprovides a number of stretches of amino acid residues (i.e. smallpeptides) that are particularly suited for binding to RANK-L. Thesestretches of amino acid residues may be present in, and/or may beincorporated into, an amino acid sequence of the invention, inparticular in such a way that they form (part of) the antigen bindingsite of an amino acid sequence of the invention. As these stretches ofamino acid residues were first generated as CDR sequences of heavy chainantibodies or V_(HH) sequences that were raised against RANK-L (or maybe based on and/or derived from such CDR sequences, as further describedherein), they will also generally be referred to herein as “CDRsequences” (i.e. as CDR1 sequences, CDR2 sequences and CDR3 sequences,respectively). It should however be noted that the invention in itsbroadest sense is not limited to a specific structural role or functionthat these stretches of amino acid residues may have in an amino acidsequence of the invention, as long as these stretches of amino acidresidues allow the amino acid sequence of the invention to bind toRANK-L. Thus, generally, the invention in its broadest sense comprisesany amino acid sequence that is capable of binding to RANK-L and thatcomprises one or more CDR sequences as described herein, and inparticular a suitable combination of two or more such CDR sequences,that are suitably linked to each other via one or more further aminoacid sequences, such that the entire amino acid sequence forms a bindingdomain and/or binding unit that is capable of binding to RANK-L. Itshould however also be noted that the presence of only one such CDRsequence in an amino acid sequence of the invention may by itselfalready be sufficient to provide an amino acid sequence of the inventionthat is capable of binding to RANK-L; reference is for example againmade to the so-called “Expedite fragments” described in WO 03/050531.

Thus, in another specific, but non-limiting aspect, the amino acidsequence of the invention may be an amino acid sequence that comprisesat least one amino acid sequence that is chosen from the groupconsisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences thatare described herein (or any suitable combination thereof). Inparticular, an amino acid sequence of the invention may be an amino acidsequence that comprises at least one antigen binding site, wherein saidantigen binding site comprises at least one amino acid sequence that ischosen from the group consisting of the CDR1 sequences, CDR2 sequencesand CDR3 sequences that are described herein (or any suitablecombination thereof).

Generally, in this aspect of the invention, the amino acid sequence ofthe invention may be any amino acid sequence that comprises at least onestretch of amino acid residues, in which said stretch of amino acidresidues has an amino acid sequence that corresponds to the sequence ofat least one of the CDR sequences described herein. Such an amino acidsequence may or may not comprise an immunoglobulin fold. For example,and without limitation, such an amino acid sequence may be a suitablefragment of an immunoglobulin sequence that comprises at least one suchCDR sequence, but that is not large enough to form a (complete)immunoglobulin fold (reference is for example again made to the“Expedite fragments” described in WO 03/050531). Alternatively, such anamino acid sequence may be a suitable “protein scaffold” that comprisesleast one stretch of amino acid residues that corresponds to such a CDRsequence (i.e. as part of its antigen binding site). Suitable scaffoldsfor presenting amino acid sequences will be clear to the skilled person,and for example comprise, without limitation, to binding scaffolds basedon or derived from immunoglobulins (i.e. other than the immunoglobulinsequences already described herein), protein scaffolds derived fromprotein A domains (such as Affibodies™), tendamistat, fibronectin,lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimersand PDZ domains (Binz et al., Nat. Biotech 2005, Vol 23:1257), andbinding moieties based on DNA or RNA including but not limited to DNA orRNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 20069(8):619-32).

Again, any amino acid sequence of the invention that comprises one ormore of these CDR sequences is preferably such that it can specificallybind (as defined herein) to RANK-L, and more in particular such that itcan bind to RANK-L with an affinity (suitably measured and/or expressedas a K_(D)-value (actual or apparent), a K_(A)-value (actual orapparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as anIC₅₀ value, as further described herein), that is as defined herein.

More in particular, the amino acid sequences according to this aspect ofthe invention may be any amino acid sequence that comprises at least oneantigen binding site, wherein said antigen binding site comprises atleast two amino acid sequences that are chosen from the group consistingof the CDR1 sequences described herein, the CDR2 sequences describedherein and the CDR3 sequences described herein, such that (i) when thefirst amino acid sequence is chosen from the CDR1 sequences describedherein, the second amino acid sequence is chosen from the CDR2 sequencesdescribed herein or the CDR3 sequences described herein; (ii) when thefirst amino acid sequence is chosen from the CDR2 sequences describedherein, the second amino acid sequence is chosen from the CDR1 sequencesdescribed herein or the CDR3 sequences described herein; or (iii) whenthe first amino acid sequence is chosen from the CDR3 sequencesdescribed herein, the second amino acid sequence is chosen from the CDR1sequences described herein or the CDR3 sequences described herein.

Even more in particular, the amino acid sequences of the invention maybe amino acid sequences that comprise at least one antigen binding site,wherein said antigen binding site comprises at least three amino acidsequences that are chosen from the group consisting of the CDR1sequences described herein, the CDR2 sequences described herein and theCDR3 sequences described herein, such that the first amino acid sequenceis chosen from the CDR1 sequences described herein, the second aminoacid sequence is chosen from the CDR2 sequences described herein, andthe third amino acid sequence is chosen from the CDR3 sequencesdescribed herein. Preferred combinations of CDR1, CDR2 and CDR3sequences will become clear from the further description herein. As willbe clear to the skilled person, such an amino acid sequence ispreferably an immunoglobulin sequence (as further described herein), butit may for example also be any other amino acid sequence that comprisesa suitable scaffold for presenting said CDR sequences.

Thus, in one specific, but non-limiting aspect, the invention relates toan amino acid sequence directed against RANK-L, that comprises one ormore stretches of amino acid residues chosen from the group consistingof:

-   a) the amino acid sequences of SEQ ID NO's: 188-249;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;-   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    312-373 and 758;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    312-373 and 758;-   g) the amino acid sequences of SEQ ID NO's: 436-497;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    436-497;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    436-497;    or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more aminoacid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence    according to b) and/or c) is preferably, and compared to the    corresponding amino acid sequence according to a), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to b) and/or c) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to a);    and/or-   iii) the amino acid sequence according to b) and/or c) may be an    amino acid sequence that is derived from an amino acid sequence    according to a) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence    according to e) and/or f) is preferably, and compared to the    corresponding amino acid sequence according to d), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to e) and/or f) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to d);    and/or-   iii) the amino acid sequence according to e) and/or f) may be an    amino acid sequence that is derived from an amino acid sequence    according to d) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence    according to h) and/or i) is preferably, and compared to the    corresponding amino acid sequence according to g), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to h) and/or i) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to g);    and/or-   iii) the amino acid sequence according to h) and/or i) may be an    amino acid sequence that is derived from an amino acid sequence    according to g) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any amino acid sequences of the invention thatcomprise one or more amino acid sequences according to b), c), e), f),h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

-   i) the amino acid sequences of SEQ ID NO's: 188-249;-   ii) the amino acid sequences of SEQ ID NO's: 312-373 and 758; and-   iii) the amino acid sequences of SEQ ID NO's: 436-497;    or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against RANK-L.

In a more specific, but again non-limiting aspect, the invention relatesto an amino acid sequence directed against RANK-L, that comprises two ormore stretches of amino acid residues chosen from the group consistingof:

-   a) the amino acid sequences of SEQ ID NO's: 188-249;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;-   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    312-373 and 758;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    312-373 and 758;-   g) the amino acid sequences of SEQ ID NO's: 436-497;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    436-497;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    436-497;    such that (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences according to a), b)    or c), the second stretch of amino acid residues corresponds to one    of the amino acid sequences according to d), e), f), g), h) or    i); (ii) when the first stretch of amino acid residues corresponds    to one of the amino acid sequences according to d), e) or f), the    second stretch of amino acid residues corresponds to one of the    amino acid sequences according to a), b), c), g), h) or i); or (iii)    when the first stretch of amino acid residues corresponds to one of    the amino acid sequences according to g), h) or i), the second    stretch of amino acid residues corresponds to one of the amino acid    sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

-   i) the amino acid sequences of SEQ ID NO's: 188-249;-   ii) the amino acid sequences of SEQ ID NO's: 312-373 and 758; and-   iii) the amino acid sequences of SEQ ID NO's: 436-497;    such that, (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's:    188-249, the second stretch of amino acid residues corresponds to    one of the amino acid sequences of SEQ ID NO's: 312-373 and 758 or    of SEQ ID NO's: 436-497; (ii) when the first stretch of amino acid    residues corresponds to one of the amino acid sequences of SEQ ID    NO's: 312-373 and 758, the second stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's:    188-249 or of SEQ ID NO's: 436-497; or (iii) when the first stretch    of amino acid residues corresponds to one of the amino acid    sequences of SEQ ID NO's: 436-497, the second stretch of amino acid    residues corresponds to one of the amino acid sequences of SEQ ID    NO's: 188-249 or of SEQ ID NO's: 312-373 and 758.

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against RANK-L.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against RANK-L, that comprises threeor more stretches of amino acid residues, in which the first stretch ofamino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 188-249;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;    the second stretch of amino acid residues is chosen from the group    consisting of:-   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    312-373 and 758;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    312-373 and 758;    and the third stretch of amino acid residues is chosen from the    group consisting of:-   g) the amino acid sequences of SEQ ID NO's: 436-497;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    436-497;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    436-497.

Preferably, in this specific aspect, the first stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 188-249; the second stretch of amino acid residues ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 312-373 and 758; and the third stretch of amino acid residues ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 436-497.

Again, preferably, in such an amino acid sequence, the at least threestretches of amino acid residues forms part of the antigen binding sitefor binding against RANK-L.

Preferred combinations of such stretches of amino acid sequences willbecome clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 560-621. This degree of amino acid identity can for example bedetermined by determining the degree of amino acid identity (in a mannerdescribed herein) between said amino acid sequence and one or more ofthe sequences of SEQ ID NO's: 560-621, in which the amino acid residuesthat form the framework regions are disregarded. Also, such amino acidsequences of the invention can be as further described herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to RANK-L; and more in particularbind to RANK-L with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), the amino acidsequence of the invention is preferably such that:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 188-249;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;    and/or

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    312-373 and 758;-   amino acid sequences that have 3, 2, or 1 amino acid difference with    at least one of the amino acid sequences of SEQ ID NO's: 312-373 and    758;    and/or

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 436-497;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    436-497;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    436-497.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 188-249; and/or CDR2 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 312-373 and 758;and/or CDR3 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 436-497.

In particular, when the amino acid sequence of the invention essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), theamino acid sequence of the invention is preferably such that:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 188-249;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;    and

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    312-373 and 758;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    312-373 and 758;    and

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 436-497;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    436-497;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    436-497; or any suitable fragment of such an amino acid sequence.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 188-249; and CDR2 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 312-373 and 758;and CDR3 is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 436-497.

Again, preferred combinations of CDR sequences will become clear fromthe further description herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to RANK-L; and more in particularbind to RANK-L with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which the CDR sequences of said aminoacid sequence have at least 70% amino acid identity, preferably at least80% amino acid identity, more preferably at least 90% amino acididentity, such as 95% amino acid identity or more or even essentially100% amino acid identity with the CDR sequences of at least one of theamino acid sequences of SEQ ID NO's: 560-621. This degree of amino acididentity can for example be determined by determining the degree ofamino acid identity (in a manner described herein) between said aminoacid sequence and one or more of the sequences of SEQ ID NO's: 560-621,in which the amino acid residues that form the framework regions aredisregarded. Such amino acid sequences of the invention can be asfurther described herein.

In such an amino acid sequence of the invention, the framework sequencesmay be any suitable framework sequences, and examples of suitableframework sequences will be clear to the skilled person, for example onthe basis the standard handbooks and the further disclosure and priorart mentioned herein.

The framework sequences are preferably (a suitable combination of)immunoglobulin framework sequences or framework sequences that have beenderived from immunoglobulin framework sequences (for example, byhumanization or camelization). For example, the framework sequences maybe framework sequences derived from a light chain variable domain (e.g.a V_(L)-sequence) and/or from a heavy chain variable domain (e.g. aV_(H)-sequence). In one particularly preferred aspect, the frameworksequences are either framework sequences that have been derived from aV_(HH)-sequence (in which said framework sequences may optionally havebeen partially or fully humanized) or are conventional V_(H) sequencesthat have been camelized (as defined herein).

The framework sequences are preferably such that the amino acid sequenceof the invention is a domain antibody (or an amino acid sequence that issuitable for use as a domain antibody); is a single domain antibody (oran amino acid sequence that is suitable for use as a single domainantibody); is a “dAb” (or an amino acid sequence that is suitable foruse as a dAb); or is a NANOBODY™ (including but not limited to V_(HH)sequence). Again, suitable framework sequences will be clear to theskilled person, for example on the basis the standard handbooks and thefurther disclosure and prior art mentioned herein.

In particular, the framework sequences present in the amino acidsequences of the invention may contain one or more of Hallmark residues(as defined herein), such that the amino acid sequence of the inventionis a NANOBODY. Some preferred, but non-limiting examples of (suitablecombinations of) such framework sequences will become clear from thefurther disclosure herein.

Again, as generally described herein for the amino acid sequences of theinvention, it is also possible to use suitable fragments (orcombinations of fragments) of any of the foregoing, such as fragmentsthat contain one or more CDR sequences, suitably flanked by and/orlinked via one or more framework sequences (for example, in the sameorder as these CDR's and framework sequences may occur in the full-sizedimmunoglobulin sequence from which the fragment has been derived). Suchfragments may also again be such that they comprise or can form animmunoglobulin fold, or alternatively be such that they do not compriseor cannot form an immunoglobulin fold.

In one specific aspect, such a fragment comprises a single CDR sequenceas described herein (and in particular a CDR3 sequence), that is flankedon each side by (part of) a framework sequence (and in particular, partof the framework sequence(s) that, in the immunoglobulin sequence fromwhich the fragment is derived, are adjacent to said CDR sequence. Forexample, a CDR3 sequence may be preceded by (part of) a FR3 sequence andfollowed by (part of) a FR4 sequence). Such a fragment may also containa disulphide bridge, and in particular a disulphide bridge that linksthe two framework regions that precede and follow the CDR sequence,respectively (for the purpose of forming such a disulphide bridge,cysteine residues that naturally occur in said framework regions may beused, or alternatively cysteine residues may be synthetically added toor introduced into said framework regions). For a further description ofthese “Expedite fragments”, reference is again made to WO 03/050531, aswell as to the US provisional application of Ablynx N.V. entitled“Peptides capable of binding to serum proteins” of Ablynx N.V.(inventors: Revets, Hilde Adi Pierrette; Kolkman, Joost Alexander; andHoogenboom, Hendricus Renerus Jacobus Mattheus) filed on Dec. 5, 2006(see also PCT/EP2007/063348).

In another aspect, the invention relates to a compound or construct, andin particular a protein or polypeptide (also referred to herein as a“compound of the invention” or “polypeptide of the invention”,respectively) that comprises or essentially consists of one or moreamino acid sequences of the invention (or suitable fragments thereof),and optionally further comprises one or more other groups, residues,moieties or binding units. As will become clear to the skilled personfrom the further disclosure herein, such further groups, residues,moieties, binding units or amino acid sequences may or may not providefurther functionality to the amino acid sequence of the invention(and/or to the compound or construct in which it is present) and may ormay not modify the properties of the amino acid sequence of theinvention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional amino acid sequences, such that thecompound or construct is a (fusion) protein or (fusion) polypeptide. Ina preferred but non-limiting aspect, said one or more other groups,residues, moieties or binding units are immunoglobulin sequences. Evenmore preferably, said one or more other groups, residues, moieties orbinding units are chosen from the group consisting of domain antibodies,amino acid sequences that are suitable for use as a domain antibody,single domain antibodies, amino acid sequences that are suitable for useas a single domain antibody, “dAb”'s, amino acid sequences that aresuitable for use as a dAb, or NANOBODIES.

Alternatively, such groups, residues, moieties or binding units may forexample be chemical groups, residues, moieties, which may or may not bythemselves be biologically and/or pharmacologically active. For example,and without limitation, such groups may be linked to the one or moreamino acid sequences of the invention so as to provide a “derivative” ofan amino acid sequence or polypeptide of the invention, as furtherdescribed herein.

Also within the scope of the present invention are compounds orconstructs, that comprises or essentially consists of one or morederivatives as described herein, and optionally further comprises one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are amino acid sequences.

In the compounds or constructs described above, the one or more aminoacid sequences of the invention and the one or more groups, residues,moieties or binding units may be linked directly to each other and/orvia one or more suitable linkers or spacers. For example, when the oneor more groups, residues, moieties or binding units are amino acidsequences, the linkers may also be amino acid sequences, so that theresulting compound or construct is a fusion (protein) or fusion(polypeptide).

The compounds or polypeptides of the invention can generally be preparedby a method which comprises at least one step of suitably linking theone or more amino acid sequences of the invention to the one or morefurther groups, residues, moieties or binding units, optionally via theone or more suitable linkers, so as to provide the compound orpolypeptide of the invention. Polypeptides of the invention can also beprepared by a method which generally comprises at least the steps ofproviding a nucleic acid that encodes a polypeptide of the invention,expressing said nucleic acid in a suitable manner, and recovering theexpressed polypeptide of the invention. Such methods can be performed ina manner known per se, which will be clear to the skilled person, forexample on the basis of the methods and techniques further describedherein.

The process of designing/selecting and/or preparing a compound orpolypeptide of the invention, starting from an amino acid sequence ofthe invention, is also referred to herein as “formatting” said aminoacid sequence of the invention; and an amino acid of the invention thatis made part of a compound or polypeptide of the invention is said to be“formatted” or to be “in the format of” said compound or polypeptide ofthe invention. Examples of ways in which an amino acid sequence of theinvention can be formatted and examples of such formats will be clear tothe skilled person based on the disclosure herein; and such formattedamino acid sequences form a further aspect of the invention.

In one specific aspect of the invention, a compound of the invention ora polypeptide of the invention may have an increased half-life, comparedto the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such compounds and polypeptideswill become clear to the skilled person based on the further disclosureherein, and for example comprise amino acid sequences or polypeptides ofthe invention that have been chemically modified to increase thehalf-life thereof (for example, by means of pegylation); amino acidsequences of the invention that comprise at least one additional bindingsite for binding to a serum protein (such as serum albumin); orpolypeptides of the invention that comprise at least one amino acidsequence of the invention that is linked to at least one moiety (and inparticular at least one amino acid sequence) that increases thehalf-life of the amino acid sequence of the invention. Examples ofpolypeptides of the invention that comprise such half-life extendingmoieties or amino acid sequences will become clear to the skilled personbased on the further disclosure herein; and for example include, withoutlimitation, polypeptides in which the one or more amino acid sequencesof the invention are suitable linked to one or more serum proteins orfragments thereof (such as (human) serum albumin or suitable fragmentsthereof) or to one or more binding units that can bind to serum proteins(such as, for example, domain antibodies, amino acid sequences that aresuitable for use as a domain antibody, single domain antibodies, aminoacid sequences that are suitable for use as a single domain antibody,“dAb”'s, amino acid sequences that are suitable for use as a dAb, orNANOBODIES that can bind to serum proteins such as serum albumin (suchas human serum albumin), serum immunoglobulins such as IgG, ortransferrin; reference is made to the further description and referencesmentioned herein); polypeptides in which an amino acid sequence of theinvention is linked to an Fc portion (such as a human Fc) or a suitablepart or fragment thereof; or polypeptides in which the one or more aminoacid sequences of the invention are suitable linked to one or more smallproteins or peptides that can bind to serum proteins (such as, withoutlimitation, the proteins and peptides described in WO 91/01743, WO01/45746, WO 02/076489 and to the US provisional application of AblynxN.V. entitled “Peptides capable of binding to serum proteins” of AblynxN.V. filed on Dec. 5, 2006 (see also PCT/EP2007/063348).

Generally, the compounds or polypeptides of the invention with increasedhalf-life preferably have a half-life that is at least 1.5 times,preferably at least 2 times, such as at least 5 times, for example atleast 10 times or more than 20 times, greater than the half-life of thecorresponding amino acid sequence of the invention per se. For example,the compounds or polypeptides of the invention with increased half-lifemay have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such compoundsor polypeptides of the invention have a serum half-life that isincreased with more than 1 hours, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24, 48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, suchcompounds or polypeptides of the invention exhibit a serum half-life inhuman of at least about 12 hours, preferably at least 24 hours, morepreferably at least 48 hours, even more preferably at least 72 hours ormore. For example, compounds or polypeptides of the invention may have ahalf-life of at least 5 days (such as about 5 to 10 days), preferably atleast 9 days (such as about 9 to 14 days), more preferably at leastabout 10 days (such as about 10 to 15 days), or at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 18 days or more), or more than 14 days (such asabout 14 to 19 days).

In another aspect, the invention relates to a nucleic acid that encodesan amino acid sequence of the invention or a polypeptide of theinvention (or a suitable fragment thereof). Such a nucleic acid willalso be referred to herein as a “nucleic acid of the invention” and mayfor example be in the form of a genetic construct, as further describedherein.

In another aspect, the invention relates to a host or host cell thatexpresses (or that under suitable circumstances is capable ofexpressing) an amino acid sequence of the invention and/or a polypeptideof the invention; and/or that contains a nucleic acid of the invention.Some preferred but non-limiting examples of such hosts or host cellswill become clear from the further description herein.

The invention further relates to a product or composition containing orcomprising at least one amino acid sequence of the invention, at leastone polypeptide of the invention (or a suitable fragment thereof) and/orat least one nucleic acid of the invention, and optionally one or morefurther components of such compositions known per se, i.e. depending onthe intended use of the composition. Such a product or composition mayfor example be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or compositions will become clear from the furtherdescription herein.

The invention also relates to the use of an amino acid sequence,NANOBODY or polypeptide of the invention, or of a composition comprisingthe same, in (methods or compositions for) modulating RANK-L, either invitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an asingle cell or in a multicellular organism, and in particular in amammal, and more in particular in a human being, such as in a humanbeing that is at risk of or suffers from a bone disease or disorder).

The invention also relates to methods for modulating RANK-L, either invitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an asingle cell or multicellular organism, and in particular in a mammal,and more in particular in a human being, such as in a human being thatis at risk of or suffers from a bone disease or disorder), which methodcomprises at least the step of contacting RANK-L with at least one aminoacid sequence, NANOBODY or polypeptide of the invention, or with acomposition comprising the same, in a manner and in an amount suitableto modulate RANK-L, with at least one amino acid sequence, NANOBODY orpolypeptide of the invention.

The invention also relates to the use of an one amino acid sequence,NANOBODY or polypeptide of the invention in the preparation of acomposition (such as, without limitation, a pharmaceutical compositionor preparation as further described herein) for modulating RANK-L,either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g.in an a single cell or multicellular organism, and in particular in amammal, and more in particular in a human being, such as in a humanbeing that is at risk of or suffers from a bone disease or disorder).

In the context of the present invention, “modulating” or “to modulate”generally means either reducing or inhibiting the activity of, oralternatively increasing the activity of, RANK-L, as measured using asuitable in vitro, cellular or in vivo assay (such as those mentionedherein). In particular, “modulating” or “to modulate” may mean eitherreducing or inhibiting the activity of, or alternatively increasing theactivity of RANK-L, as measured using a suitable in vitro, cellular orin vivo assay (such as those mentioned herein), by at least 1%,preferably at least 5%, such as at least 10% or at least 25%, forexample by at least 50%, at least 60%, at least 70%, at least 80%, or90% or more, compared to activity of RANK-L in the same assay under thesame conditions but without the presence of the amino acid sequence,NANOBODY or polypeptide of the invention.

As will be clear to the skilled person, “modulating” may also involveeffecting a change (which may either be an increase or a decrease) inaffinity, avidity, specificity and/or selectivity of RANK-L for one ormore of its targets, ligands or substrates; and/or effecting a change(which may either be an increase or a decrease) in the sensitivity ofRANK-L for one or more conditions in the medium or surroundings in whichRANK-L is present (such as pH, ion strength, the presence of co-factors,etc.), compared to the same conditions but without the presence of theamino acid sequence, NANOBODY or polypeptide of the invention. As willbe clear to the skilled person, this may again be determined in anysuitable manner and/or using any suitable assay known per se, such asthe assays described herein or in the prior art cited herein.

“Modulating” may also mean effecting a change (i.e. an activity as anagonist or as an antagonist, respectively) with respect to one or morebiological or physiological mechanisms, effects, responses, functions,pathways or activities in which RANK-L (or in which its substrate(s),ligand(s) or pathway(s) are involved, such as its signalling pathway ormetabolic pathway and their associated biological or physiologicaleffects) is involved. Again, as will be clear to the skilled person,such an action as an agonist or an antagonist may be determined in anysuitable manner and/or using any suitable (in vitro and usually cellularor in assay) assay known per se, such as the assays described herein orin the prior art cited herein. In particular, an action as an agonist orantagonist may be such that an intended biological or physiologicalactivity is increased or decreased, respectively, by at least 1%,preferably at least 5%, such as at least 10% or at least 25%, forexample by at least 50%, at least 60%, at least 70%, at least 80%, or90% or more, compared to the biological or physiological activity in thesame assay under the same conditions but without the presence of theamino acid sequence, NANOBODY or polypeptide of the invention.

Modulating may for example involve reducing or inhibiting the binding ofRANK-L to one of its substrates or ligands and/or competing with anatural ligand, substrate for binding to RANK-L. Modulating may alsoinvolve activating RANK-L or the mechanism or pathway in which it isinvolved. Modulating may be reversible or irreversible, but forpharmaceutical and pharmacological purposes will usually be in areversible manner.

The invention further relates to methods for preparing or generating theamino acid sequences, polypeptides, nucleic acids, host cells, productsand compositions described herein. Some preferred but non-limitingexamples of such methods will become clear from the further descriptionherein.

Generally, these methods may comprise the steps of:

-   a) providing a set, collection or library of amino acid sequences;    and-   b) screening said set, collection or library of amino acid sequences    for amino acid sequences that can bind to and/or have affinity for    RANK-L;    and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for RANK-L.

In such a method, the set, collection or library of amino acid sequencesmay be any suitable set, collection or library of amino acid sequences.For example, the set, collection or library of amino acid sequences maybe a set, collection or library of immunoglobulin sequences (asdescribed herein), such as a naïve set, collection or library ofimmunoglobulin sequences; a synthetic or semi-synthetic set, collectionor library of immunoglobulin sequences; and/or a set, collection orlibrary of immunoglobulin sequences that have been subjected to affinitymaturation.

Also, in such a method, the set, collection or library of amino acidsequences may be a set, collection or library of heavy chain variabledomains (such as V_(H) domains or V_(HH) domains) or of light chainvariable domains. For example, the set, collection or library of aminoacid sequences may be a set, collection or library of domain antibodiesor single domain antibodies, or may be a set, collection or library ofamino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofimmunoglobulin sequences, for example derived from a mammal that hasbeen suitably immunized with RANK-L or with a suitable antigenicdeterminant based thereon or derived therefrom, such as an antigenicpart, fragment, region, domain, loop or other epitope thereof. In oneparticular aspect, said antigenic determinant may be an extracellularpart, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of amino acidsequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) amino acid sequences will beclear to the person skilled in the art, for example on the basis of thefurther disclosure herein. Reference is also made to the review byHoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating amino acid sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells expressing amino acid    sequences;-   b) screening said collection or sample of cells for cells that    express an amino acid sequence that can bind to and/or have affinity    for RANK-L;    and-   c) either (i) isolating said amino acid sequence; or (ii) isolating    from said cell a nucleic acid sequence that encodes said amino acid    sequence, followed by expressing said amino acid sequence.

For example, when the desired amino acid sequence is an immunoglobulinsequence, the collection or sample of cells may for example be acollection or sample of B-cells. Also, in this method, the sample ofcells may be derived from a mammal that has been suitably immunized withRANK-L or with a suitable antigenic determinant based thereon or derivedtherefrom, such as an antigenic part, fragment, region, domain, loop orother epitope thereof. In one particular aspect, said antigenicdeterminant may be an extracellular part, region, domain, loop or otherextracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820 (2001).

In another aspect, the method for generating an amino acid sequencedirected against RANK-L may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for RANK-L;    and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

In such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with RANK-L or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, for example on the basis of the further disclosure herein.Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating an amino acid sequencedirected against RANK-L may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for RANK-L and that is    cross-blocked or is cross blocking a NANOBODY of the invention, e.g.    SEQ ID NO's: 560-621, or a humanized NANOBODY of the invention, e.g.    SEQ ID NO's: 730-757 and 765, or a polypeptide or construct of the    invention, e.g. SEQ ID NO's: 622-729, 759-762 and 766-789; and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

The invention also relates to amino acid sequences that are obtained bythe above methods, or alternatively by a method that comprises the oneof the above methods and in addition at least the steps of determiningthe nucleotide sequence or amino acid sequence of said immunoglobulinsequence; and of expressing or synthesizing said amino acid sequence ina manner known per se, such as by expression in a suitable host cell orhost organism or by chemical synthesis.

Also, following the steps above, one or more amino acid sequences of theinvention may be suitably humanized (or alternatively camelized); and/orthe amino acid sequence(s) thus obtained may be linked to each other orto one or more other suitable amino acid sequences (optionally via oneor more suitable linkers) so as to provide a polypeptide of theinvention. Also, a nucleic acid sequence encoding an amino acid sequenceof the invention may be suitably humanized (or alternatively camelized)and suitably expressed; and/or one or more nucleic acid sequencesencoding an amino acid sequence of the invention may be linked to eachother or to one or more nucleic acid sequences that encode othersuitable amino acid sequences (optionally via nucleotide sequences thatencode one or more suitable linkers), after which the nucleotidesequence thus obtained may be suitably expressed so as to provide apolypeptide of the invention.

The invention further relates to applications and uses of the amino acidsequences, compounds, constructs, polypeptides, nucleic acids, hostcells, products and compositions described herein, as well as to methodsfor the prevention and/or treatment for diseases and disordersassociated with RANK-L. Some preferred but non-limiting applications anduses will become clear from the further description herein.

The invention also relates to the amino acid sequences, compounds,constructs, polypeptides, nucleic acids, host cells, products andcompositions described herein for use in therapy.

In particular, the invention also relates to the amino acid sequences,compounds, constructs, polypeptides, nucleic acids, host cells, productsand compositions described herein for use in therapy of a disease ordisorder that can be prevented or treated by administering, to a subjectin need thereof, of (a pharmaceutically effective amount of) an aminoacid sequence, compound, construct or polypeptide as described herein.

More in particular, the invention relates to the amino acid sequences,compounds, constructs, polypeptides, nucleic acids, host cells, productsand compositions described herein for use in therapy of bone diseasesand disorders.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description herein, in which theinvention will be described and discussed in more detail with referenceto the NANOBODIES of the invention and polypeptides of the inventioncomprising the same, which form some of the preferred aspects of theinvention.

As will become clear from the further description herein, NANOBODIESgenerally offer certain advantages (outlined herein) compared to “dAb's”or similar (single) domain antibodies or immunoglobulin sequences, whichadvantages are also provided by the NANOBODIES of the invention.However, it will be clear to the skilled person that the more generalaspects of the teaching below can also be applied (either directly oranalogously) to other amino acid sequences of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the present description, examples and claims:

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks, such as    Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd. Ed.),    Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et    al, eds., “Current protocols in molecular biology”, Green Publishing    and Wiley Interscience, New York (1987); Lewin, “Genes II”, John    Wiley & Sons, New York, N.Y., (1985); Old et al., “Principles of    Gene Manipulation: An Introduction to Genetic Engineering”, 2nd    edition, University of California Press, Berkeley, Calif. (1981);    Roitt et al., “Immunology” (6th. Ed.), Mosby/Elsevier, Edinburgh    (2001); Roitt et al., Roitt's Essential Immunology, 10^(th) Ed.    Blackwell Publishing, U K (2001); and Janeway et al.,    “Immunobiology” (6th Ed.), Garland Science Publishing/Churchill    Livingstone, New York (2005), as well as to the general background    art cited herein; b) Unless indicated otherwise, the term    “immunoglobulin sequence”—whether used herein to refer to a heavy    chain antibody or to a conventional 4-chain antibody—is used as a    general term to include both the full-size antibody, the individual    chains thereof, as well as all parts, domains or fragments thereof    (including but not limited to antigen-binding domains or fragments    such as V_(HH) domains or V_(H)/V_(L) domains, respectively). In    addition, the term “sequence” as used herein (for example in terms    like “immunoglobulin sequence”, “antibody sequence”, “variable    domain sequence”, “V_(HH) sequence” or “protein sequence”), should    generally be understood to include both the relevant amino acid    sequence as well as nucleic acid sequences or nucleotide sequences    encoding the same, unless the context requires a more limited    interpretation; c) Unless indicated otherwise, all methods, steps,    techniques and manipulations that are not specifically described in    detail can be performed and have been performed in a manner known    per se, as will be clear to the skilled person. Reference is for    example again made to the standard handbooks and the general    background art mentioned herein and to the further references cited    therein; as well as to for example the following reviews Presta,    Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56; Levin and Weiss, Mol.    Biosyst. 2006, 2(1): 49-57; Irving et al., J. Immunol. Methods,    2001, 248(1-2), 31-45; Schmitz et al., Placenta, 2000, 21 Suppl. A,    5106-12, Gonzales et al., Tumour Biol., 2005, 26(1), 31-43, which    describe techniques for protein engineering, such as affinity    maturation and other techniques for improving the specificity and    other desired properties of proteins such as immunoglobulins.-   d) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code, as mentioned in Table    A-2;

TABLE A-2 one-letter and three-letter amino acid code Nonpolar, AlanineAla A uncharged Valine Val V (at pH 6.0-7.0)⁽³⁾ Leucine Leu L IsoleucineIle I Phenylalanine Phe F Methionine⁽¹⁾ Met M Tryptophan Trp W ProlinePro P Polar, Glycine⁽²⁾ Gly G uncharged Serine Ser S (at pH 6.0-7.0)Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q TyrosineTyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH 6.0-7.0)Histidine⁽⁴⁾ His H Aspartate Asp D Glutamate Glu E Notes: ⁽¹⁾Sometimesalso considered to be a polar uncharged amino acid. ⁽²⁾Sometimes alsoconsidered to be a nonpolar uncharged amino acid. ⁽³⁾As will be clear tothe skilled person, the fact that an amino acid residue is referred toin this Table as being either charged or uncharged at pH 6.0 to 7.0 doesnot reflect in any way on the charge said amino acid residue may have ata pH lower than 6.0 and/or at a pH higher than 7.0; the amino acidresidues mentioned in the Table can be either charged and/or unchargedat such a higher or lower pH, as will be clear to the skilled person.⁽⁴⁾As is known in the art, the charge of a His residue is greatlydependant upon even small shifts in pH, but a His residue can generallybe considered essentially uncharged at a pH of about 6.5.

-   e) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated by    dividing [the number of nucleotides in the first nucleotide sequence    that are identical to the nucleotides at the corresponding positions    in the second nucleotide sequence] by [the total number of    nucleotides in the first nucleotide sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of a nucleotide in the second nucleotide sequence—compared to the    first nucleotide sequence—is considered as a difference at a single    nucleotide (position).    -   Alternatively, the degree of sequence identity between two or        more nucleotide sequences may be calculated using a known        computer algorithm for sequence alignment such as NCBI Blast        v2.0, using standard settings.    -   Some other techniques, computer algorithms and settings for        determining the degree of sequence identity are for example        described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO        00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A.    -   Usually, for the purpose of determining the percentage of        “sequence identity” between two nucleotide sequences in        accordance with the calculation method outlined hereinabove, the        nucleotide sequence with the greatest number of nucleotides will        be taken as the “first” nucleotide sequence, and the other        nucleotide sequence will be taken as the “second” nucleotide        sequence;-   f) For the purposes of comparing two or more amino acid sequences,    the percentage of “sequence identity” between a first amino acid    sequence and a second amino acid sequence (also referred to herein    as “amino acid identity”) may be calculated by dividing [the number    of amino acid residues in the first amino acid sequence that are    identical to the amino acid residues at the corresponding positions    in the second amino acid sequence] by [the total number of amino    acid residues in the first amino acid sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of an amino acid residue in the second amino acid sequence—compared    to the first amino acid sequence—is considered as a difference at a    single amino acid residue (position), i.e. as an “amino acid    difference” as defined herein.    -   Alternatively, the degree of sequence identity between two amino        acid sequences may be calculated using a known computer        algorithm, such as those mentioned above for determining the        degree of sequence identity for nucleotide sequences, again        using standard settings.    -   Usually, for the purpose of determining the percentage of        “sequence identity” between two amino acid sequences in        accordance with the calculation method outlined hereinabove, the        amino acid sequence with the greatest number of amino acid        residues will be taken as the “first” amino acid sequence, and        the other amino acid sequence will be taken as the “second”        amino acid sequence.    -   Also, in determining the degree of sequence identity between two        amino acid sequences, the skilled person may take into account        so-called “conservative” amino acid substitutions, which can        generally be described as amino acid substitutions in which an        amino acid residue is replaced with another amino acid residue        of similar chemical structure and which has little or        essentially no influence on the function, activity or other        biological properties of the polypeptide. Such conservative        amino acid substitutions are well known in the art, for example        from WO 04/037999, GB-A-3 357 768, WO 98/49185, WO 00/46383 and        WO 01/09300; and (preferred) types and/or combinations of such        substitutions may be selected on the basis of the pertinent        teachings from WO 04/037999 as well as WO 98/49185 and from the        further references cited therein.    -   Such conservative substitutions preferably are substitutions in        which one amino acid within the following groups (a)-(e) is        substituted by another amino acid residue within the same        group: (a) small aliphatic, nonpolar or slightly polar residues:        Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged        residues and their (uncharged) amides: Asp, Asn, Glu and        Gln; (c) polar, positively charged residues: His, Arg and        Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val        and Cys; and (e) aromatic residues: Phe, Tyr and Trp.    -   Particularly preferred conservative substitutions are as        follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or        into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into        Asp; Gly into Ala or into Pro; His into Asn or into Gln; He into        Leu or into Val; Leu into Ile or into Val; Lys into Arg, into        Gln or into Glu; Met into Leu, into Tyr or into He; Phe into        Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into        Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.    -   Any amino acid substitutions applied to the polypeptides        described herein may also be based on the analysis of the        frequencies of amino acid variations between homologous proteins        of different species developed by Schulz et al., Principles of        Protein Structure, Springer-Verlag, 1978, on the analyses of        structure forming potentials developed by Chou and Fasman,        Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978,        and on the analysis of hydrophobicity patterns in proteins        developed by Eisenberg et al., Proc. Natl. Acad Sci. USA 81:        140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157: 105-132,        198 1, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353,        1986, all incorporated herein in their entirety by reference.        Information on the primary, secondary and tertiary structure of        NANOBODIES is given in the description herein and in the general        background art cited above. Also, for this purpose, the crystal        structure of a V_(HH) domain from a llama is for example given        by Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803        (1996); Spinelli et al., Natural Structural Biology (1996); 3,        752-757; and Decanniere et al., Structure, Vol. 7, 4, 361        (1999). Further information about some of the amino acid        residues that in conventional V_(H) domains form the V_(H)/V_(L)        interface and potential camelizing substitutions on these        positions can be found in the prior art cited above.-   g) Amino acid sequences and nucleic acid sequences are said to be    “exactly the same” if they have 100% sequence identity (as defined    herein) over their entire length;-   h) When comparing two amino acid sequences, the term “amino acid    difference” refers to an insertion, deletion or substitution of a    single amino acid residue on a position of the first sequence,    compared to the second sequence; it being understood that two amino    acid sequences can contain one, two or more such amino acid    differences;-   i) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this may mean that the latter    nucleotide sequence or amino acid sequence has been incorporated    into the firstmentioned nucleotide sequence or amino acid sequence,    respectively, but more usually this generally means that the    firstmentioned nucleotide sequence or amino acid sequence comprises    within its sequence a stretch of nucleotides or amino acid residues,    respectively, that has the same nucleotide sequence or amino acid    sequence, respectively, as the latter sequence, irrespective of how    the firstmentioned sequence has actually been generated or obtained    (which may for example be by any suitable method described herein).    By means of a non-limiting example, when a NANOBODY of the invention    is said to comprise a CDR sequence, this may mean that said CDR    sequence has been incorporated into the NANOBODY of the invention,    but more usually this generally means that the NANOBODY of the    invention contains within its sequence a stretch of amino acid    residues with the same amino acid sequence as said CDR sequence,    irrespective of how said NANOBODY of the invention has been    generated or obtained. It should also be noted that when the latter    amino acid sequence has a specific biological or structural    function, it preferably has essentially the same, a similar or an    equivalent biological or structural function in the firstmentioned    amino acid sequence (in other words, the firstmentioned amino acid    sequence is preferably such that the latter sequence is capable of    performing essentially the same, a similar or an equivalent    biological or structural function). For example, when a NANOBODY of    the invention is said to comprise a CDR sequence or framework    sequence, respectively, the CDR sequence and framework are    preferably capable, in said NANOBODY, of functioning as a CDR    sequence or framework sequence, respectively. Also, when a    nucleotide sequence is said to comprise another nucleotide sequence,    the firstmentioned nucleotide sequence is preferably such that, when    it is expressed into an expression product (e.g. a polypeptide), the    amino acid sequence encoded by the latter nucleotide sequence forms    part of said expression product (in other words, that the latter    nucleotide sequence is in the same reading frame as the    firstmentioned, larger nucleotide sequence).-   j) A nucleic acid sequence or amino acid sequence is considered to    be “(in) essentially isolated (form)”—for example, compared to its    native biological source and/or the reaction medium or cultivation    medium from which it has been obtained—when it has been separated    from at least one other component with which it is usually    associated in said source or medium, such as another nucleic acid,    another protein/polypeptide, another biological component or    macromolecule or at least one contaminant, impurity or minor    component. In particular, a nucleic acid sequence or amino acid    sequence is considered “essentially isolated” when it has been    purified at least 2-fold, in particular at least 10-fold, more in    particular at least 100-fold, and up to 1000-fold or more. A nucleic    acid sequence or amino acid sequence that is “in essentially    isolated form” is preferably essentially homogeneous, as determined    using a suitable technique, such as a suitable chromatographical    technique, such as polyacrylamide-gel electrophoresis;-   k) The term “domain” as used herein generally refers to a globular    region of an amino acid sequence (such as an antibody chain, and in    particular to a globular region of a heavy chain antibody), or to a    polypeptide that essentially consists of such a globular region.    Usually, such a domain will comprise peptide loops (for example 3 or    4 peptide loops) stabilized, for example, as a sheet or by disulfide    bonds. The term “binding domain” refers to such a domain that is    directed against an antigenic determinant (as defined herein);-   l) The term “antigenic determinant” refers to the epitope on the    antigen recognized by the antigen-binding molecule (such as a    NANOBODY or a polypeptide of the invention) and more in particular    by the antigen-binding site of said molecule. The terms “antigenic    determinant” and “epitope” may also be used interchangeably herein.-   m) An amino acid sequence (such as a NANOBODY, an antibody, a    polypeptide of the invention, or generally an antigen binding    protein or polypeptide or a fragment thereof) that can    (specifically) bind to, that has affinity for and/or that has    specificity for a specific antigenic determinant, epitope, antigen    or protein (or for at least one part, fragment or epitope thereof)    is said to be “against” or “directed against” said antigenic    determinant, epitope, antigen or protein.-   n) The term “specificity” refers to the number of different types of    antigens or antigenic determinants to which a particular    antigen-binding molecule or antigen-binding protein (such as a    NANOBODY or a polypeptide of the invention) molecule can bind. The    specificity of an antigen-binding protein can be determined based on    affinity and/or avidity. The affinity, represented by the    equilibrium constant for the dissociation of an antigen with an    antigen-binding protein (K_(D)), is a measure for the binding    strength between an antigenic determinant and an antigen-binding    site on the antigen-binding protein: the lesser the value of the    K_(D), the stronger the binding strength between an antigenic    determinant and the antigen-binding molecule (alternatively, the    affinity can also be expressed as the affinity constant (K_(A)),    which is 1/K_(D)). As will be clear to the skilled person (for    example on the basis of the further disclosure herein), affinity can    be determined in a manner known per se, depending on the specific    antigen of interest. Avidity is the measure of the strength of    binding between an antigen-binding molecule (such as a NANOBODY or    polypeptide of the invention) and the pertinent antigen. Avidity is    related to both the affinity between an antigenic determinant and    its antigen binding site on the antigen-binding molecule and the    number of pertinent binding sites present on the antigen-binding    molecule. Typically, antigen-binding proteins (such as the amino    acid sequences, NANOBODIES and/or polypeptides of the invention)    will bind to their antigen with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²    moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter    (i.e. with an association constant (K_(A)) of 10⁵ to 10¹²    liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or more    and more preferably 10⁸ to 10¹² liter/moles). Any K_(D) value    greater than 10⁴ mol/liter (or any K_(A) value lower than 10⁴ M⁻¹)    liters/mol is generally considered to indicate non-specific binding.    Preferably, a monovalent immunoglobulin sequence of the invention    will bind to the desired antigen with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM. Specific binding of an antigen-binding protein    to an antigen or antigenic determinant can be determined in any    suitable manner known per se, including, for example, Scatchard    analysis and/or competitive binding assays, such as    radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich    competition assays, and the different variants thereof known per se    in the art; as well as the other techniques mentioned herein.    -   The dissociation constant may be the actual or apparent        dissociation constant, as will be clear to the skilled person.        Methods for determining the dissociation constant will be clear        to the skilled person, and for example include the techniques        mentioned herein. In this respect, it will also be clear that it        may not be possible to measure dissociation constants of more        then 10⁻⁴ moles/liter or 10⁻³ moles/liter (e.g., of 10⁻²        moles/liter). Optionally, as will also be clear to the skilled        person, the (actual or apparent) dissociation constant may be        calculated on the basis of the (actual or apparent) association        constant (K_(A)), by means of the relationship [K_(D)=1/K_(A)].    -   The affinity denotes the strength or stability of a molecular        interaction. The affinity is commonly given as by the K_(D), or        dissociation constant, which has units of mol/liter (or M). The        affinity can also be expressed as an association constant,        K_(A), which equals 1/K_(D) and has units of (mol/liter)⁻¹ (or        M⁻¹). In the present specification, the stability of the        interaction between two molecules (such as an amino acid        sequence, NANOBODY or polypeptide of the invention and its        intended target) will mainly be expressed in terms of the K_(D)        value of their interaction; it being clear to the skilled person        that in view of the relation K_(A)=1/K_(D), specifying the        strength of molecular interaction by its K_(D) value can also be        used to calculate the corresponding K_(A) value. The K_(D)-value        characterizes the strength of a molecular interaction also in a        thermodynamic sense as it is related to the free energy (DG) of        binding by the well known relation DG=RT·ln(K_(D)) (equivalently        DG=-RT·ln(K_(A))), where R equals the gas constant, T equals the        absolute temperature and ln denotes the natural logarithm.    -   The K_(D) for biological interactions which are considered        meaningful (e.g. specific) are typically in the range of 10⁻¹° M        (0.1 nM) to 10⁻⁵M (10000 nM). The stronger an interaction is,        the lower is its K_(D).    -   The K_(D) can also be expressed as the ratio of the dissociation        rate constant of a complex, denoted as k_(off), to the rate of        its association, denoted k_(on) (so that K_(D)=k_(off)/k_(on)        and K_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹        (where s is the SI unit notation of second). The on-rate k_(on)        has units M⁻¹s⁻¹. The on-rate may vary between 10² M⁻¹s⁻¹ to        about 10⁷ M⁻¹s⁻¹, approaching the diffusion-limited association        rate constant for bimolecular interactions. The off-rate is        related to the half-life of a given molecular interaction by the        relation t_(1/2)=ln(2)/k_(off). The off-rate may vary between        10⁻⁶ s⁻¹ (near irreversible complex with a t_(1/2) of multiple        days) to 1 s⁻¹ (t_(1/2)=0.69 s).    -   The affinity of a molecular interaction between two molecules        can be measured via different techniques known per se, such as        the well known surface plasmon resonance (SPR) biosensor        technique (see for example Ober et al., Intern Immunology, 13,        1551-1559, 2001) where one molecule is immobilized on the        biosensor chip and the other molecule is passed over the        immobilized molecule under flow conditions yielding k_(on),        k_(off) measurements and hence K_(D) (or K_(A)) values. This can        for example be performed using the well-known BIACORE        instruments.    -   It will also be clear to the skilled person that the measured        K_(D) may correspond to the apparent K_(D) if the measuring        process somehow influences the intrinsic binding affinity of the        implied molecules for example by artefacts related to the        coating on the biosensor of one molecule. Also, an apparent        K_(D) may be measured if one molecule contains more than one        recognition sites for the other molecule. In such situation the        measured affinity may be affected by the avidity of the        interaction by the two molecules.    -   Another approach that may be used to assess affinity is the        2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of        Friguet et al. (J. Immunol. Methods, 77, 305-19, 1985). This        method establishes a solution phase binding equilibrium        measurement and avoids possible artefacts relating to adsorption        of one of the molecules on a support such as plastic.    -   However, the accurate measurement of K_(D) may be quite        labor-intensive and as consequence, often apparent K_(D) values        are determined to assess the binding strength of two molecules.        It should be noted that as long all measurements are made in a        consistent way (e.g. keeping the assay conditions unchanged)        apparent K_(D) measurements can be used as an approximation of        the true K_(D) and hence in the present document K_(D) and        apparent K_(D) should be treated with equal importance or        relevance. Finally, it should be noted that in many situations        the experienced scientist may judge it to be convenient to        determine the binding affinity relative to some reference        molecule. For example, to assess the binding strength between        molecules A and B, one may e.g. use a reference molecule C that        is known to bind to B and that is suitably labelled with a        fluorophore or chromophore group or other chemical moiety, such        as biotin for easy detection in an ELISA or FACS (Fluorescent        activated cell sorting) or other format (the fluorophore for        fluorescence detection, the chromophore for light absorption        detection, the biotin for streptavidin-mediated ELISA        detection). Typically, the reference molecule C is kept at a        fixed concentration and the concentration of A is varied for a        given concentration or amount of B. As a result an IC₅₀ value is        obtained corresponding to the concentration of A at which the        signal measured for C in absence of A is halved. Provided        K_(D ref), the K_(D) of the reference molecule, is known, as        well as the total concentration c_(ref) of the reference        molecule, the apparent K_(D) for the interaction A-B can be        obtained from following formula:        K_(D)=IC₅₀/(1+c_(ref)/K_(D ref)). Note that if        c_(ref)<<K_(D ref), K_(D)≈IC₅₀. Provided the measurement of the        IC₅₀ is performed in a consistent way (e.g. keeping c_(ref)        fixed) for the binders that are compared, the strength or        stability of a molecular interaction can be assessed by the IC₅₀        and this measurement is judged as equivalent to K_(D) or to        apparent K_(D) throughout this text.-   o) The half-life of an amino acid sequence, compound or polypeptide    of the invention can generally be defined as the time taken for the    serum concentration of the amino acid sequence, compound or    polypeptide to be reduced by 50%, in vivo, for example due to    degradation of the sequence or compound and/or clearance or    sequestration of the sequence or compound by natural mechanisms. The    in vivo half-life of an amino acid sequence, compound or polypeptide    of the invention can be determined in any manner known per se, such    as by pharmacokinetic analysis. Suitable techniques will be clear to    the person skilled in the art, and may for example generally involve    the steps of suitably administering to a warm-blooded animal (i.e.    to a human or to another suitable mammal, such as a mouse, rabbit,    rat, pig, dog or a primate, for example monkeys from the genus    Macaca (such as, and in particular, cynomolgus monkeys (Macaca    fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon    (Papio ursinus)) a suitable dose of the amino acid sequence,    compound or polypeptide of the invention; collecting blood samples    or other samples from said animal; determining the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention in said blood sample; and calculating, from (a plot    of) the data thus obtained, the time until the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention has been reduced by 50% compared to the initial level    upon dosing. Reference is for example made to the Experimental Part    below, as well as to the standard handbooks, such as Kenneth, A et    al: Chemical Stability of Pharmaceuticals: A Handbook for    Pharmacists and Peters et al, Pharmacokinete analysis: A Practical    Approach (1996). Reference is also made to “Pharmacokinetics”, M    Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition    (1982).    -   As will also be clear to the skilled person (see for example        pages 6 and 7 of WO 04/003019 and in the further references        cited therein), the half-life can be expressed using parameters        such as the t½-alpha, t½-beta and the area under the curve        (AUC). In the present specification, an “increase in half-life”        refers to an increase in any one of these parameters, such as        any two of these parameters, or essentially all three these        parameters. As used herein “increase in half-life” or “increased        half-life” in particular refers to an increase in the t½-beta,        either with or without an increase in the t½-alpha and/or the        AUC or both.-   p) In the context of the present invention, “modulating” or “to    modulate” generally means either reducing or inhibiting the activity    of, or alternatively increasing the activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay. In particular, “modulating” or “to modulate” may mean either    reducing or inhibiting the activity of, or alternatively increasing    a (relevant or intended) biological activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay (which will usually depend on the target or antigen involved),    by at least 1%, preferably at least 5%, such as at least 10% or at    least 25%, for example by at least 50%, at least 60%, at least 70%,    at least 80%, or 90% or more, compared to activity of the target or    antigen in the same assay under the same conditions but without the    presence of the construct of the invention.    -   As will be clear to the skilled person, “modulating” may also        involve effecting a change (which may either be an increase or a        decrease) in affinity, avidity, specificity and/or selectivity        of a target or antigen for one or more of its ligands, binding        partners, partners for association into a homomultimeric or        heteromultimeric form, or substrates; and/or effecting a change        (which may either be an increase or a decrease) in the        sensitivity of the target or antigen for one or more conditions        in the medium or surroundings in which the target or antigen is        present (such as pH, ion strength, the presence of co-factors,        etc.), compared to the same conditions but without the presence        of the construct of the invention. As will be clear to the        skilled person, this may again be determined in any suitable        manner and/or using any suitable assay known per se, depending        on the target or antigen involved.    -   “Modulating” may also mean effecting a change (i.e. an activity        as an agonist, as an antagonist or as a reverse agonist,        respectively, depending on the target or antigen and the desired        biological or physiological effect) with respect to one or more        biological or physiological mechanisms, effects, responses,        functions, pathways or activities in which the target or antigen        (or in which its substrate(s), ligand(s) or pathway(s) are        involved, such as its signalling pathway or metabolic pathway        and their associated biological or physiological effects) is        involved. Again, as will be clear to the skilled person, such an        action as an agonist or an antagonist may be determined in any        suitable manner and/or using any suitable (in vitro and usually        cellular or in assay) assay known per se, depending on the        target or antigen involved. In particular, an action as an        agonist or antagonist may be such that an intended biological or        physiological activity is increased or decreased, respectively,        by at least 1%, preferably at least 5%, such as at least 10% or        at least 25%, for example by at least 50%, at least 60%, at        least 70%, at least 80%, or 90% or more, compared to the        biological or physiological activity in the same assay under the        same conditions but without the presence of the construct of the        invention.    -   Modulating may for example also involve allosteric modulation of        the target or antigen; and/or reducing or inhibiting the binding        of the target or antigen to one of its substrates or ligands        and/or competing with a natural ligand, substrate for binding to        the target or antigen. Modulating may also involve activating        the target or antigen or the mechanism or pathway in which it is        involved. Modulating may for example also involve effecting a        change in respect of the folding or confirmation of the target        or antigen, or in respect of the ability of the target or        antigen to fold, to change its confirmation (for example, upon        binding of a ligand), to associate with other (sub)units, or to        disassociate. Modulating may for example also involve effecting        a change in the ability of the target or antigen to transport        other compounds or to serve as a channel for other compounds        (such as ions).    -   Modulating may be reversible or irreversible, but for        pharmaceutical and pharmacological purposes will usually be in a        reversible manner-   q) In respect of a target or antigen, the term “interaction site” on    the target or antigen means a site, epitope, antigenic determinant,    part, domain or stretch of amino acid residues on the target or    antigen that is a site for binding to a ligand, receptor or other    binding partner, a catalytic site, a cleavage site, a site for    allosteric interaction, a site involved in multimerisation (such as    homomerization or heterodimerization) of the target or antigen; or    any other site, epitope, antigenic determinant, part, domain or    stretch of amino acid residues on the target or antigen that is    involved in a biological action or mechanism of the target or    antigen. More generally, an “interaction site” can be any site,    epitope, antigenic determinant, part, domain or stretch of amino    acid residues on the target or antigen to which an amino acid    sequence or polypeptide of the invention can bind such that the    target or antigen (and/or any pathway, interaction, signalling,    biological mechanism or biological effect in which the target or    antigen is involved) is modulated (as defined herein).-   r) An amino acid sequence or polypeptide is said to be “specific    for” a first target or antigen compared to a second target or    antigen when is binds to the first antigen with an affinity (as    described above, and suitably expressed as a K_(D) value, K_(A)    value, K_(off) rate and/or K_(on) rate) that is at least 10 times,    such as at least 100 times, and preferably at least 1000 times, and    up to 10.000 times or more better than the affinity with which said    amino acid sequence or polypeptide binds to the second target or    polypeptide. For example, the first antigen may bind to the target    or antigen with a K_(D) value that is at least 10 times less, such    as at least 100 times less, and preferably at least 1000 times less,    such as 10.000 times less or even less than that, than the K_(D)    with which said amino acid sequence or polypeptide binds to the    second target or polypeptide. Preferably, when an amino acid    sequence or polypeptide is “specific for” a first target or antigen    compared to a second target or antigen, it is directed against (as    defined herein) said first target or antigen, but not directed    against said second target or antigen.-   s) The terms “cross-block”, “cross-blocked” and “cross-blocking” are    used interchangeably herein to mean the ability of an amino acid    sequence or other binding agents (such as a polypeptide of the    invention) to interfere with the binding of other amino acid    sequences or binding agents of the invention to a given target. The    extend to which an amino acid sequence or other binding agent of the    invention is able to interfere with the binding of another to    RANK-L, and therefore whether it can be said to cross-block    according to the invention, can be determined using competition    binding assays. One particularly suitable quantitative    cross-blocking assay uses a BIACORE machine which can measure the    extent of interactions using surface plasmon resonance technology.    Another suitable quantitative cross-blocking assay uses an    ELISA-based approach to measure competition between amino acid    sequences or other binding agents in terms of their binding to the    target.    -   The following generally describes a suitable BIACORE assay for        determining whether an amino acid sequence or other binding        agent cross-blocks or is capable of cross-blocking according to        the invention. It will be appreciated that the assay can be used        with any of the amino acid sequence or other binding agents        described herein. The BIACORE machine (for example the        BIACORE 3000) is operated in line with the manufacturer's        recommendations. Thus in one cross-blocking assay, the target        protein is coupled to a CM5 BIACORE chip using standard amine        coupling chemistry to generate a surface that is coated with the        target. Typically 200-800 resonance units of the target would be        coupled to the chip (an amount that gives easily measurable        levels of binding but that is readily saturable by the        concentrations of test reagent being used). Two test amino acid        sequences (termed A* and B*) to be assessed for their ability to        cross-block each other are mixed at a one to one molar ratio of        binding sites in a suitable buffer to create the test mixture.        When calculating the concentrations on a binding site basis the        molecular weight of an amino acid sequence is assumed to be the        total molecular weight of the amino acid sequence divided by the        number of target binding sites on that amino acid sequence. The        concentration of each amino acid sequence in the test mix should        be high enough to readily saturate the binding sites for that        amino acid sequence on the target molecules captured on the        BIACORE chip. The amino acid sequences in the mixture are at the        same molar concentration (on a binding basis) and that        concentration would typically be between 1.00 and 1.5 micromolar        (on a binding site basis). Separate solutions containing A*        alone and B* alone are also prepared. A* and B* in these        solutions should be in the same buffer and at the same        concentration as in the test mix. The test mixture is passed        over the target-coated BIACORE chip and the total amount of        binding recorded. The chip is then treated in such a way as to        remove the bound amino acid sequences without damaging the        chip-bound target. Typically this is done by treating the chip        with 30 mM HCl for 60 seconds. The solution of A* alone is then        passed over the target-coated surface and the amount of binding        recorded. The chip is again treated to remove all of the bound        amino acid sequences without damaging the chip-bound target. The        solution of B* alone is then passed over the target-coated        surface and the amount of binding recorded. The maximum        theoretical binding of the mixture of A* and B* is next        calculated, and is the sum of the binding of each amino acid        sequence when passed over the target surface alone. If the        actual recorded binding of the mixture is less than this        theoretical maximum then the two amino acid sequences are        cross-blocking each other. Thus, in general, a cross-blocking        amino acid sequence or other binding agent according to the        invention is one which will bind to the target in the above        BIACORE cross-blocking assay such that during the assay and in        the presence of a second amino acid sequence or other binding        agent of the invention the recorded binding is between 80% and        0.1% (e.g. 80% to 4%) of the maximum theoretical binding,        specifically between 75% and 0.1% (e.g. 75% to 4%) of the        maximum theoretical binding, and more specifically between 70%        and 0.1% (e.g. 70% to 4%) of maximum theoretical binding (as        just defined above) of the two amino acid sequences or binding        agents in combination. The BIACORE assay described above is a        primary assay used to determine if amino acid sequences or other        binding agents cross-block each other according to the        invention. On rare occasions particular amino acid sequences or        other binding agents may not bind to target coupled via amine        chemistry to a CM5 BIACORE chip (this usually occurs when the        relevant binding site on target is masked or destroyed by the        coupling to the chip). In such cases cross-blocking can be        determined using a tagged version of the target, for example a        N-terminal His-tagged version. In this particular format, an        anti-His amino acid sequence would be coupled to the BIACORE        chip and then the His-tagged target would be passed over the        surface of the chip and captured by the anti-His amino acid        sequence. The cross blocking analysis would be carried out        essentially as described above, except that after each chip        regeneration cycle, new His-tagged target would be loaded back        onto the anti-His amino acid sequence coated surface. In        addition to the example given using N-terminal His-tagged        target, C-terminal His-tagged target could alternatively be        used. Furthermore, various other tags and tag binding protein        combinations that are known in the art could be used for such a        cross-blocking analysis (e.g. HA tag with anti-HA antibodies;        FLAG tag with anti-FLAG antibodies; biotin tag with        streptavidin).    -   The following generally describes an ELISA assay for determining        whether an amino acid sequence or other binding agent directed        against a target cross-blocks or is capable of cross-blocking as        defined herein. It will be appreciated that the assay can be        used with any of the amino acid sequences (or other binding        agents such as polypeptides of the invention) described herein.        The general principal of the assay is to have an amino acid        sequence or binding agent that is directed against the target        coated onto the wells of an ELISA plate. An excess amount of a        second, potentially cross-blocking, anti-target amino acid        sequence is added in solution (i.e. not bound to the ELISA        plate). A limited amount of the target is then added to the        wells. The coated amino acid sequence and the amino acid        sequence in solution compete for binding of the limited number        of target molecules. The plate is washed to remove excess target        that has not been bound by the coated amino acid sequence and to        also remove the second, solution phase amino acid sequence as        well as any complexes formed between the second, solution phase        amino acid sequence and target. The amount of bound target is        then measured using a reagent that is appropriate to detect the        target. An amino acid sequence in solution that is able to        cross-block the coated amino acid sequence will be able to cause        a decrease in the number of target molecules that the coated        amino acid sequence can bind relative to the number of target        molecules that the coated amino acid sequence can bind in the        absence of the second, solution phase, amino acid sequence. In        the instance where the first amino acid sequence, e.g. an Ab-X,        is chosen to be the immobilized amino acid sequence, it is        coated onto the wells of the ELISA plate, after which the plates        are blocked with a suitable blocking solution to minimize        non-specific binding of reagents that are subsequently added. An        excess amount of the second amino acid sequence, i.e. Ab-Y, is        then added to the ELISA plate such that the moles of Ab-Y target        binding sites per well are at least 10 fold higher than the        moles of Ab-X target binding sites that were used, per well,        during the coating of the ELISA plate. Target is then added such        that the moles of target added per well are at least 25-fold        lower than the moles of Ab-X target binding sites that were used        for coating each well. Following a suitable incubation period        the ELISA plate is washed and a reagent for detecting the target        is added to measure the amount of target specifically bound by        the coated anti-target amino acid sequence (in this case Ab-X).        The background signal for the assay is defined as the signal        obtained in wells with the coated amino acid sequence (in this        case Ab-X), second solution phase amino acid sequence (in this        case Ab-Y), target buffer only (i.e. without target added) and        target detection reagents. The positive control signal for the        assay is defined as the signal obtained in wells with the coated        amino acid sequence (in this case Ab-X), second solution phase        amino acid sequence buffer only (i.e. without second solution        phase amino acid sequence added), target and target detection        reagents. The ELISA assay may be run in such a manner so as to        have the positive control signal be at least 6 times the        background signal. To avoid any artefacts (e.g. significantly        different affinities between Ab-X and Ab-Y for the target)        resulting from the choice of which amino acid sequence to use as        the coating amino acid sequence and which to use as the second        (competitor) amino acid sequence, the cross-blocking assay may        to be run in two formats: 1) format 1 is where Ab-X is the amino        acid sequence that is coated onto the ELISA plate and Ab-Y is        the competitor amino acid sequence that is in solution and 2)        format 2 is where Ab-Y is the amino acid sequence that is coated        onto the ELISA plate and Ab-X is the competitor amino acid        sequence that is in solution. Ab-X and Ab-Y are defined as        cross-blocking if, either in format 1 or in format 2, the        solution phase anti-target amino acid sequence is able to cause        a reduction of between 60% and 100%, specifically between 70%        and 100%, and more specifically between 80% and 100%, of the        target detection signal {i.e. the amount of target bound by the        coated amino acid sequence) as compared to the target detection        signal obtained in the absence of the solution phase anti-target        amino acid sequence (i.e. the positive control wells).-   t) An amino acid sequence is said to be “cross-reactive” for two    different antigens or antigenic determinants if it is specific for    (as defined herein) both these different antigens or antigenic    determinants.-   u) By binding that is “essentially independent of the pH” is    generally meant herein that the association constant (K_(A)) of the    amino acid sequence with respect to the serum protein (such as serum    albumin) at the pH value(s) that occur in a cell of an animal or    human body (as further described herein) is at least 5%, such as at    least 10%, preferably at least 25%, more preferably at least 50%,    even more preferably at least 60%, such as even more preferably at    least 70%, such as at least 80% or 90% or more (or even more than    100%, such as more than 110%, more than 120% or even 130% or more,    or even more than 150%, or even more than 200%) of the association    constant (K_(A)) of the amino acid sequence with respect to the same    serum protein at the pH value(s) that occur outside said cell.    Alternatively, by binding that is “essentially independent of the    pH” is generally meant herein that the k_(off) rate (measured by    BIACORE) of the amino acid sequence with respect to the serum    protein (such as serum albumin) at the pH value(s) that occur in a    cell of an animal or human body (as e.g. further described herein,    e.g. pH around 5.5, e.g. 5.3 to 5.7) is at least 5%, such as at    least 10%, preferably at least 25%, more preferably at least 50%,    even more preferably at least 60%, such as even more preferably at    least 70%, such as at least 80% or 90% or more (or even more than    100%, such as more than 110%, more than 120% or even 130% or more,    or even more than 150%, or even more than 200%) of the k_(off) rate    of the amino acid sequence with respect to the same serum protein at    the pH value(s) that occur outside said cell, e.g. pH 7.2 to 7.4. By    “the pH value(s) that occur in a cell of an animal or human body” is    meant the pH value(s) that may occur inside a cell, and in    particular inside a cell that is involved in the recycling of the    serum protein. In particular, by “the pH value(s) that occur in a    cell of an animal or human body” is meant the pH value(s) that may    occur inside a (sub)cellular compartment or vesicle that is involved    in recycling of the serum protein (e.g. as a result of pinocytosis,    endocytosis, transcytosis, exocytosis and phagocytosis or a similar    mechanism of uptake or internalization into said cell), such as an    endosome, lysosome or pinosome.-   v) As further described herein, the total number of amino acid    residues in a NANOBODY can be in the region of 110-120, is    preferably 112-115, and is most preferably 113. It should however be    noted that parts, fragments, analogs or derivatives (as further    described herein) of a NANOBODY are not particularly limited as to    their length and/or size, as long as such parts, fragments, analogs    or derivatives meet the further requirements outlined herein and are    also preferably suitable for the purposes described herein;-   w) The amino acid residues of a NANOBODY are numbered according to    the general numbering for V_(H) domains given by Kabat et al.    (“Sequence of proteins of immunological interest”, US Public Health    Services, NIH Bethesda, Md., Publication No. 91), as applied to    V_(HH) domains from Camelids in the article of Riechmann and    Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195    (see for example FIG. 2 of this publication); or referred to herein.    According to this numbering, FR1 of a NANOBODY comprises the amino    acid residues at positions 1-30, CDR1 of a NANOBODY comprises the    amino acid residues at positions 31-35, FR2 of a NANOBODY comprises    the amino acids at positions 36-49, CDR2 of a NANOBODY comprises the    amino acid residues at positions 50-65, FR3 of a NANOBODY comprises    the amino acid residues at positions 66-94, CDR3 of a NANOBODY    comprises the amino acid residues at positions 95-102, and FR4 of a    NANOBODY comprises the amino acid residues at positions 103-113. [In    this respect, it should be noted that—as is well known in the art    for V_(H) domains and for V_(HH) domains—the total number of amino    acid residues in each of the CDR's may vary and may not correspond    to the total number of amino acid residues indicated by the Kabat    numbering (that is, one or more positions according to the Kabat    numbering may not be occupied in the actual sequence, or the actual    sequence may contain more amino acid residues than the number    allowed for by the Kabat numbering). This means that, generally, the    numbering according to Kabat may or may not correspond to the actual    numbering of the amino acid residues in the actual sequence.    Generally, however, it can be said that, according to the numbering    of Kabat and irrespective of the number of amino acid residues in    the CDR's, position 1 according to the Kabat numbering corresponds    to the start of FR1 and vice versa, position 36 according to the    Kabat numbering corresponds to the start of FR2 and vice versa,    position 66 according to the Kabat numbering corresponds to the    start of FR3 and vice versa, and position 103 according to the Kabat    numbering corresponds to the start of FR4 and vice versa.].    -   Alternative methods for numbering the amino acid residues of        V_(H) domains, which methods can also be applied in an analogous        manner to V_(HH) domains from Camelids and to NANOBODIES, are        the method described by Chothia et al. (Nature 342, 877-883        (1989)), the so-called “AbM definition” and the so-called        “contact definition”. However, in the present description,        claims and figures, the numbering according to Kabat as applied        to V_(HH) domains by Riechmann and Muyldermans will be followed,        unless indicated otherwise; and-   x) The Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the prior art citedherein, to the review article by Muyldermans in Reviews in MolecularBiotechnology 74(2001), 277-302; as well as to the following patentapplications, which are mentioned as general background art: WO94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel;WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 ofthe Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 ofAlgonomics N.V. and Ablynx N.V.; WO 01/90190 by the National ResearchCouncil of Canada; WO 03/025020 (=EP 1 433 793) by the Institute ofAntibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the furtherpublished patent applications by Ablynx N.V. Reference is also made tothe further prior art mentioned in these applications, and in particularto the list of references mentioned on pages 41-43 of the Internationalapplication WO 06/040153, which list and references are incorporatedherein by reference.

In accordance with the terminology used in the art (see the abovereferences), the variable domains present in naturally occurring heavychain antibodies will also be referred to as “V_(HH) domains”, in orderto distinguish them from the heavy chain variable domains that arepresent in conventional 4-chain antibodies (which will be referred tohereinbelow as “V_(H) domains”) and from the light chain variabledomains that are present in conventional 4-chain antibodies (which willbe referred to hereinbelow as “V_(L) domains”).

As mentioned in the prior art referred to above, V_(HH) domains have anumber of unique structural characteristics and functional propertieswhich make isolated V_(HH) domains (as well as NANOBODIES based thereon,which share these structural characteristics and functional propertieswith the naturally occurring V_(HH) domains) and proteins containing thesame highly advantageous for use as functional antigen-binding domainsor proteins. In particular, and without being limited thereto, V_(HH)domains (which have been “designed” by nature to functionally bind to anantigen without the presence of, and without any interaction with, alight chain variable domain) and NANOBODIES can function as a single,relatively small, functional antigen-binding structural unit, domain orprotein. This distinguishes the V_(HH) domains from the V_(H) and V_(L)domains of conventional 4-chain antibodies, which by themselves aregenerally not suited for practical application as single antigen-bindingproteins or domains, but need to be combined in some form or another toprovide a functional antigen-binding unit (as in for exampleconventional antibody fragments such as Fab fragments; in ScFv'sfragments, which consist of a V_(H) domain covalently linked to a V_(L)domain).

Because of these unique properties, the use of V_(HH) domains andNANOBODIES as single antigen-binding proteins or as antigen-bindingdomains (i.e. as part of a larger protein or polypeptide) offers anumber of significant advantages over the use of conventional V_(H) andV_(L) domains, scFv's or conventional antibody fragments (such as Fab-or F(ab′)₂-fragments):

-   -   only a single domain is required to bind an antigen with high        affinity and with high selectivity, so that there is no need to        have two separate domains present, nor to assure that these two        domains are present in the right spatial conformation and        configuration (i.e. through the use of especially designed        linkers, as with scFv's);    -   V_(HH) domains and NANOBODIES can be expressed from a single        gene and require no post-translational folding or modifications;    -   V_(HH) domains and NANOBODIES can easily be engineered into        multivalent and multispecific formats (as further discussed        herein);    -   V_(HH) domains and NANOBODIES are highly soluble and do not have        a tendency to aggregate (as with the mouse-derived “dAb's”        described by Ward et al., Nature, Vol. 341, 1989, p. 544);    -   V_(HH) domains and NANOBODIES are highly stable to heat, pH,        proteases and other denaturing agents or conditions (see for        example Ewert et al, supra);    -   V_(HH) domains and NANOBODIES are easy and relatively cheap to        prepare, even on a scale required for production. For example,        V_(HH) domains, NANOBODIES and proteins/polypeptides containing        the same can be produced using microbial fermentation (e.g. as        further described below) and do not require the use of mammalian        expression systems, as with for example conventional antibody        fragments;    -   V_(HH) domains and NANOBODIES are relatively small        (approximately 15 kDa, or 10 times smaller than a conventional        IgG) compared to conventional 4-chain antibodies and        antigen-binding fragments thereof, and therefore show high(er)        penetration into tissues (including but not limited to solid        tumors and other dense tissues) than such conventional 4-chain        antibodies and antigen-binding fragments thereof;    -   V_(HH) domains and NANOBODIES can show so-called cavity-binding        properties (inter alia due to their extended CDR3 loop, compared        to conventional V_(H) domains) and can therefore also access        targets and epitopes not accessible to conventional 4-chain        antibodies and antigen-binding fragments thereof. For example,        it has been shown that V_(HH) domains and NANOBODIES can inhibit        enzymes (see for example WO 97/49805; Transue et al., Proteins        1998 Sep. 1; 32(4): 515-22; Lauwereys et al., EMBO J. 1998 Jul.        1; 17(13): 3512-20).

In a specific and preferred aspect, the invention provides NANOBODIESagainst RANK-L, and in particular NANOBODIES against RANK-L from awarm-blooded animal, and more in particular NANOBODIES against RANK-Lfrom a mammal, and especially NANOBODIES against human RANK-L; as wellas proteins and/or polypeptides comprising at least one such NANOBODY.

In particular, the invention provides NANOBODIES against RANK-L, andproteins and/or polypeptides comprising the same, that have improvedtherapeutic and/or pharmacological properties and/or other advantageousproperties (such as, for example, improved ease of preparation and/orreduced costs of goods), compared to conventional antibodies againstRANK-L or fragments thereof, compared to constructs that could be basedon such conventional antibodies or antibody fragments (such as Fab′fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies” and othermultispecific constructs (see for example the review by Holliger andHudson, Nat Biotechnol. 2005 September; 23(9):1126-36)), and alsocompared to the so-called “dAb's” or similar (single) domain antibodiesthat may be derived from variable domains of conventional antibodies.These improved and advantageous properties will become clear from thefurther description herein, and for example include, without limitation,one or more of:

-   -   increased affinity and/or avidity for RANK-L, either in a        monovalent format, in a multivalent format (for example in a        bivalent format) and/or in a multispecific format (for example        one of the multispecific formats described hereinbelow);    -   better suitability for formatting in a multivalent format (for        example in a bivalent format);    -   better suitability for formatting in a multispecific format (for        example one of the multispecific formats described hereinbelow);    -   improved suitability or susceptibility for “humanizing”        substitutions (as defined herein);    -   less immunogenicity, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described hereinbelow);    -   increased stability, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described hereinbelow);    -   increased specificity towards RANK-L, either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described hereinbelow);    -   decreased or where desired increased cross-reactivity with        RANK-L from different species;        and/or    -   one or more other improved properties desirable for        pharmaceutical use (including prophylactic use and/or        therapeutic use) and/or for diagnostic use (including but not        limited to use for imaging purposes), either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described hereinbelow).

As generally described herein for the amino acid sequences of theinvention, the NANOBODIES of the invention are preferably in essentiallyisolated form (as defined herein), or form part of a protein orpolypeptide of the invention (as defined herein), which may comprise oressentially consist of one or more NANOBODIES of the invention and whichmay optionally further comprise one or more further amino acid sequences(all optionally linked via one or more suitable linkers). For example,and without limitation, the one or more amino acid sequences of theinvention may be used as a binding unit in such a protein orpolypeptide, which may optionally contain one or more further amino acidsequences that can serve as a binding unit (i.e. against one or moreother targets than RANK-L), so as to provide a monovalent, multivalentor multispecific polypeptide of the invention, respectively, all asdescribed herein. In particular, such a protein or polypeptide maycomprise or essentially consist of one or more NANOBODIES of theinvention and optionally one or more (other) NANOBODIES (i.e. directedagainst other targets than RANK-L), all optionally linked via one ormore suitable linkers, so as to provide a monovalent, multivalent ormultispecific NANOBODY construct, respectively, as further describedherein. Such proteins or polypeptides may also be in essentiallyisolated form (as defined herein).

In a NANOBODY of the invention, the binding site for binding againstRANK-L is preferably formed by the CDR sequences. Optionally, a NANOBODYof the invention may also, and in addition to the at least one bindingsite for binding against RANK-L, contain one or more further bindingsites for binding against other antigens, proteins or targets. Formethods and positions for introducing such second binding sites,reference is for example made to Keck and Huston, Biophysical Journal,71, October 1996, 2002-2011; EP 0 640 130 and WO 06/07260.

As generally described herein for the amino acid sequences of theinvention, when a NANOBODY of the invention (or a polypeptide of theinvention comprising the same) is intended for administration to asubject (for example for therapeutic and/or diagnostic purposes asdescribed herein), it is preferably directed against human RANK-L;whereas for veterinary purposes, it is preferably directed againstRANK-L from the species to be treated. Also, as with the amino acidsequences of the invention, a NANOBODY of the invention may or may notbe cross-reactive (i.e. directed against RANK-L from two or more speciesof mammal, such as against human RANK-L and RANK-L from at least one ofthe species of mammal mentioned herein).

Also, again as generally described herein for the amino acid sequencesof the invention, the NANOBODIES of the invention may generally bedirected against any antigenic determinant, epitope, part, domain,subunit or confirmation (where applicable) of RANK-L. However, it isgenerally assumed and preferred that the NANOBODIES of the invention(and polypeptides comprising the same) are directed against the bindingsite for RANK on RANK-L or the binding site for OPG on RANK-L. Inanother aspect of the present invention, the amino acid sequences andpolypeptides of the invention are preferably directed against an epitopeon RANK-L that overlaps with the epitope of Denosumab.

As already described herein, the amino acid sequence and structure of aNANOBODY can be considered—without however being limited thereto—to becomprised of four framework regions or “FR's” (or sometimes alsoreferred to as “FW's”), which are referred to in the art and herein as“Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as“Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”,respectively; which framework regions are interrupted by threecomplementary determining regions or “CDR's”, which are referred to inthe art as “Complementarity Determining Region 1” or “CDR1”; as“Complementarity Determining Region 2” or “CDR2”; and as“Complementarity Determining Region 3” or “CDR3”, respectively. Somepreferred framework sequences and CDR's (and combinations thereof) thatare present in the NANOBODIES of the invention are as described herein.Other suitable CDR sequences can be obtained by the methods describedherein.

According to a non-limiting but preferred aspect of the invention, (theCDR sequences present in) the NANOBODIES of the invention are such that:

-   -   the NANOBODIES can bind to RANK-L with a dissociation constant        (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably        10⁻⁷ to 10⁻¹² moles/liter or less and more preferably 10⁻⁸ to        10⁻¹² moles/liter (i.e. with an association constant (K_(A)) of        10⁵ to 10¹² liter/moles or more, and preferably 10⁷ to 10¹²        liter/moles or more and more preferably 10⁸ to 10¹²        liter/moles);        and/or such that:    -   the NANOBODIES can bind to RANK-L with a k_(on)-rate of between        10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹        and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷        M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   the NANOBODIES can bind to RANK-L with a k_(off) rate between 1        s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶        s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, (the CDR sequences present in) the NANOBODIES of theinvention are such that: a monovalent NANOBODY of the invention (or apolypeptide that contains only one NANOBODY of the invention) ispreferably such that it will bind to RANK-L with an affinity less than500 nM, preferably less than 200 nM, more preferably less than 10 nM,such as less than 500 pM.

The affinity of the NANOBODY of the invention against RANK-L can bedetermined in a manner known per se, for example using the generaltechniques for measuring K_(D). K_(A), k_(off) or k_(on) mentionedherein, as well as some of the specific assays described herein.

Some preferred IC50 values for binding of the NANOBODIES of theinvention (and of polypeptides comprising the same) to RANK-L willbecome clear from the further description and examples herein.

In a preferred but non-limiting aspect, the invention relates to aNANOBODY (as defined herein) against RANK-L, which consists of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively), in which:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 188-249;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;    and/or

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    312-373 and 758;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    312-373 and 758;    and/or

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 436-497;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    436-497;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    436-497;    or any suitable fragment of such an amino acid sequence.

In particular, according to this preferred but non-limiting aspect, theinvention relates to a NANOBODY (as defined herein) against RANK-L,which consists of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 188-249;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    188-249;    and

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    312-373 and 758;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    312-373 and 758;    and

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 436-497;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    436-497;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    436-497;    or any suitable fragment of such an amino acid sequences.

As generally mentioned herein for the amino acid sequences of theinvention, when a NANOBODY of the invention contains one or more CDR1sequences according to b) and/or c):

-   i) any amino acid substitution in such a CDR according to b)    and/or c) is preferably, and compared to the corresponding CDR    according to a), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to b) and/or c) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to a);    and/or-   iii) the CDR according to b) and/or c) may be a CDR that is derived    from a CDR according to a) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Similarly, when a NANOBODY of the invention contains one or more CDR2sequences according to e) and/or f):

-   i) any amino acid substitution in such a CDR according to e)    and/or f) is preferably, and compared to the corresponding CDR    according to d), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to e) and/or f) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to d);    and/or-   iii) the CDR according to e) and/or f) may be a CDR that is derived    from a CDR according to d) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Also, similarly, when a NANOBODY of the invention contains one or moreCDR3 sequences according to h) and/or i):

-   i) any amino acid substitution in such a CDR according to h)    and/or i) is preferably, and compared to the corresponding CDR    according to g), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to h) and/or i) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to g);    and/or-   iii) the CDR according to h) and/or i) may be a CDR that is derived    from a CDR according to g) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

It should be understood that the last three paragraphs generally applyto any NANOBODY of the invention that comprises one or more CDR1sequences, CDR2 sequences and/or CDR3 sequences according to b), c), e),f), h) or i), respectively.

Of the NANOBODIES of the invention, NANOBODIES comprising one or more ofthe CDR's explicitly listed above are particularly preferred; NANOBODIEScomprising two or more of the CDR's explicitly listed above are moreparticularly preferred; and NANOBODIES comprising three of the CDR'sexplicitly listed above are most particularly preferred.

Some particularly preferred, but non-limiting combinations of CDRsequences, as well as preferred combinations of CDR sequences andframework sequences, are mentioned in Table A-1 below, which lists theCDR sequences and framework sequences that are present in a number ofpreferred (but non-limiting) NANOBODIES of the invention. As will beclear to the skilled person, a combination of CDR1, CDR2 and CDR3sequences that occur in the same clone (i.e. CDR1, CDR2 and CDR3sequences that are mentioned on the same line in Table A-1) will usuallybe preferred (although the invention in its broadest sense is notlimited thereto, and also comprises other suitable combinations of theCDR sequences mentioned in Table A-1). Also, a combination of CDRsequences and framework sequences that occur in the same clone (i.e. CDRsequences and framework sequences that are mentioned on the same line inTable A-1) will usually be preferred (although the invention in itsbroadest sense is not limited thereto, and also comprises other suitablecombinations of the CDR sequences and framework sequences mentioned inTable A-1, as well as combinations of such CDR sequences and othersuitable framework sequences, e.g. as further described herein).

Also, in the NANOBODIES of the invention that comprise the combinationsof CDR's mentioned in Table A-1, each CDR can be replaced by a CDRchosen from the group consisting of amino acid sequences that have atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 99% sequence identity (as defined herein) withthe mentioned CDR's; in which:

-   i) any amino acid substitution in such a CDR is preferably, and    compared to the corresponding CDR sequence mentioned in Table A-1, a    conservative amino acid substitution (as defined herein);    and/or-   ii) any such CDR sequence preferably only contains amino acid    substitutions, and no amino acid deletions or insertions, compared    to the corresponding CDR sequence mentioned in Table A-1;    and/or-   iii) any such CDR sequence is a CDR that is derived by means of a    technique for affinity maturation known per se, and in particular    starting from the corresponding CDR sequence mentioned in Table A-1.

However, as will be clear to the skilled person, the (combinations of)CDR sequences, as well as (the combinations of) CDR sequences andframework sequences mentioned in Table A-1 will generally be preferred.

TABLE A-1 Preferred combinations of CDR sequences, preferredcombinations of framework sequences, and preferred combinations offramework and CDR sequences. (“ID” refers to the SEQ ID NO in theattached sequence listing) Clone ID FR1 ID CDR 1 ID FR2 ID CDR 2 ID FR3ID CDR 3 ID FR4 RANKL1 126 EVQLVESGGGL 188 SSTMA 250 WFRQPPG 312SISTSGTRTL 374 RFTISRDNAKSTGYLQ 436 VNRRGWE 498 WGLGAQVTVSS VQAGGSLRLSCGERDFVA YADSVKG MNSLKPEDTAVYFCAA FWRLASG AVSGRTFS YDY RANKL2 127EVQLVESGGGL 189 SYYMS 251 WVRQAPG 313 SIYSDGSTTD 375 RFTISRDNAKNTLNLQ437 DANSGGL 499 WGQGTQVTV VQPGGSLRLSC KGLEWVS YADSVKG MNSLKSEDTAVYYCAKEYDY SS AASGFTFS RANKL3 128 EVQLVESGGKL 190 IYNMA 252 WFRQGPG 314RIYWSDDNT 376 RFTISRDNATNTVYLQ 438 KTTKWSLE 500 WGQGTQVTV VQAGGSLRLSCKGRESVG YYADSVKG MNSLKPEDTAVYYCAG YDY SS AVSGRTSS RANKL4 129 KVQLVESGGGL191 SYAMG 253 WFRQAPG 315 VINYRGSSLK 377 RFTISRDNAKNMVYLQ 439 QTSGADF501 WGQGTQVTV VQTGDSLRLSC KEREFVA YADRVKG MNSLKPDDTAVYYCAA GTTPQRY SSAASGRAIG TY RANKL5 130 EVQLVESGGGL 192 GHTMA 254 WFRQAPG 316 TITSSGSTIFY378 RFTISRDNGKKTMTLE 440 RIRGKVTV 502 WGQGTQVTV VQAGGSLRLSC KERDFVAADSVKG MDSLKPEDTAVYYCAA DNFDYAY SS AASGRTIG RANKL6 131 EVQLVESGGGL 193YYTAS 255 WFRQAPG 317 AISPSGNTYY 379 RFTISRDNGKHTMYLQ 441 RATDSIYY 503WGQGTQVTV MQTGGSLRLS KEREFVA ADSVKG MNSLNPEDTAVYFCAI ASSYRH SS CAASGVTYSRANKL7 132 EVQLVESEGGP 194 VSTIA 256 WFRQAPG 318 AIYPSGRNAY 380RFTISRDNAKKTVYLQ 442 HQPSGSY 504 WGQGTQVTV VQSGGSLRLSC EGREFVA VADSVKGMNSLKPEDTAAYYCAA YSAEAYAY SS AASGRTFS RANKL8 133 EVQLVESGGG 195 RYAMG257 WFRQAPG 319 AISVGGTYQ 381 RFTISRDNAESTVYLQ 443 DASPYGYL 505WGQGTQVTV SVQPGGSLRLS KEREFVS YYVDSVKG MNSLKPEDTAVYYCAG REYTATRF SSCAASGGTFS DY RANKL9 134 EVQLVESGGGL 196 SYAMG 258 WFRQAPG 320 AINYSGGST382 RFTISRDNAKNTLYLQ 444 GSGYASL 506 WGQGTQVTV VQAGGSLRLTC KEREFVANYADSVKG MNSLEPEDTAVYYCAA SYYSTERA SS AASGRTFR YTY RANKL10 135EVQLVESGGGL 197 SRTMG 259 WFRQAPG 321 AITPSSRTTY 383 RFTISRDNAKNTVLLQ445 ERTYGSN 507 WGQGTQVTV VQAGGSLRLSC KEREFVA YADSVKG MNSLKPEDTAVYYCAAYTRPTAW SS AASGITFS NY RANKL11 136 EVQLVESGGGL 198 SKTMG 260 WFRQPPG 322AITPTSRTTY 384 RFTISRDNAKNTVSLQ 446 VRRYGSP 508 WGQGTQVTV VQAGGSLRLSCNEREFVA YADSVKG MNSLKFEDTAAYYCVA PHDGSSY SS AASGRTFS EY RANKL12 137EVQLVESGGG 199 MA 261 WFRQAPG 323 AITGSGRSTY 385 RFTISRDNAKNTAYLQ 447LRGLGLEY 509 WGQGTQVTV WMQAGGSLRL KEREFVA YTDSVKG MKSLKPEDTAVYYCAGDSAKSYSY SS SCAASGRTFT RANKL13 138 EVQLVESGGGL 200 SYPMG 262 WFRQAPG 324SITGSGGSTY 386 RFTISRDNAKNTVYLQ 448 YIRPDTYL 510 WGQGTQVTV VQAGGSLRLSCKEREFVA YADSVKG MNSLRPEDTAVYSCAA SRDYRKY SS AASGRTFR DY RANKL14 139EVQLVESGGGL 201 YYTMS 263 WFRQDPG 325 AVPLSGNTY 387 RFTISRDNAKNTADLQ 449RASGSIYN 511 WGQGTQVTV VQAGGSLRLSC KEREFVA YADPVRG MNSLKPEDTAVYYCAARGSYAY SS AASGRTSS RANKL15 140 EVQLVESGGGL 202 NYVMG 264 WFRQAPG 326AISTGGSWT 388 RFTISRDNTKNTVYLQ 450 TTPATTYL 512 WGQGTQVTV VQAGGSLRLSCKEREFVT GYVDSVKD MASLKPEDTAVYYCAA PRSERQY SS AAAGGTFR DY RANKL16 141EVQLVESGGGL 203 SYAMG 265 WFRQVPG 327 AISTGSITIYG 389 RFTISRDNAKNTVYLQ451 GKREPYL 513 WGQGTQVTV VQAGGSLRLSC KERDFVA DSVKG MNSLKPEDTAVYYCAARQYTASN SS VASRRTFS PYDY RANKL17 142 EVQLVESGGGL 204 STYVMG 266 WFRQAPG328 AVSWSSGNA 390 RFATSRDTAKNIMYLQ 452 GRGYGLL 514 WGQGTQVTV VQVGDSLRLSCKEREFVA YYIDSAKG MNSLKPEDTAVYTCAA SEYTQAP SS EASGRSRF RYDY RANKL18 143EVQLVESGGGL 205 RSAMG 267 WFRQAPG 329 FITGSGGTTY 391 RFTISRDNAQNPVYLQ453 YRRTYISS 515 WGQGTQVTV VQAGGSLRLSC KEREFVG YGESVKG MNSLKPEDTAVYYCGVTYSESSEY SS AASGRTFS DY RANKL19 144 EVQLVESGGGL 206 MG 268 WFRQAPG 330SITGSGSVTN 392 RFTISRDNAKNTVFLQ 454 YLPSPYYS 516 WGQGTQVTV VQAGDSLRLSCKEREFVA YADSVKG MNSLKPEDTAVYYCAA SYYDSTKY SS AASGRTVT EY RANKL20 145EVQLVESGGGL 207 MG 269 WFRQAPG 331 AISGSGKITN 393 RFTISRDHAKNTVFLQ 455YLRSPYYS 517 WGQGTQVTV VQAGDSLRLSC TEREFVA YADSVKG MDSLKPEDTAVYYCAGSFYDSAKY SS AASGRTFT EY RANKL21 146 EVQLVESGGGL 208 SYAVG 270 WFRQVPG332 AISTGSVTIY 394 RFTISRDNAKNTVYLQ 456 GNREPYL 518 WGQGTQVTVVQAGGSLRLSC EERDFVA ADSVKG MNSLKPEDTAVYYCAA RQYTASN SS VASRRTFN PYDYRANKL22 147 EVQLVESGGGL 209 NYGMG 271 WFRQAPG 333 AITSAGGTTY 395RFTISRDSAKYTVYLQ 457 KLQIGGR 519 RGQGTQVTVSS MQTGGSLRLS KEREFVA YGDFVKGMNSLKPEDTAVYWCAA WHNLNDY CAASERTSR GY RANKL23 148 EVQLVESGGGL 210 VYTMA272 WFRQAPG 334 AITRSGKTTY 396 RFTISRDNAKNTVNLQ 458 KALLGMTN 520WGQETQVTV VQAGGSLRLSC KEREFVA YADSVKG MNSLKPDDTAVYYCAA PAGYEY SS AASGLTTRANKL3D4 149 EVQLVEAGGGL 211 GTMA 273 WFRQAPG 335 TVTSSGSTTF 397RFTISRDNAENTVNLQ 459 RIRGKVTP 521 WGQGTQVTV VQAGDSLRLSC KDREFVA YADSVKGMDSLKPEDTAVYYCAA SNYDYAY SS AASGRTIR RANKL 150 evqlvesgggwmq 212 MA 274wfrqasgker 336 AITGSGRSTY 398 rftisrdnakntaylqmkslkped 460 LRGLGLEY 522wgqgtqvtvss PMP4B3 aggslrlscaasgrtft efva YTDSVKG tavyycag DSAKSYSYRANKL 151 EVQLVESGGGL 213 SYPMG 275 WFRQAPG 337 SITGSGGSTY 399RFTISRDNAKNTVYLQ 461 YIRPDTYL 523 WGQGTQVTV PMP2E11 VQAGGSLRLSC KEREFVAYADSVKG MNSLRPEDTAVYSCAA SRDYRKY SS AASGRTFR DY RANKL 152evqlvesggglvqag 214 RYTMS 276 wfrqdpgker 338 AVPLSGNTY 400rftisrdnakntvdlqmnslkpe 462 RASGSIFN 524 wgqgtqvtvss PMP2A6gslrlscaasgltss efva YADPVRG dtavyycaa RGSYAY RANKL 153 evqlvesggglvpag215 RYTMS 277 wfrqdpgker 339 AVPLSGNTY 401 rftisrdnakntvdlqmnslkpe 463RASGSIFN 525 wgqgtqvtvss PMP1F2 gslrlscaasgltdr efva YADPVRG dtavyycaaRGSYAY RANKL 154 evqlvesggglvpag 216 RYTMS 278 wfrqdpgker 340 AVPLSGNTY402 rftisrdnakntvdlqmnslkpe 464 RASGSIFN 526 wgqgtqvtvss PMP2D4gslrlscaasgltdr efva YADPVRG dtavyycaa RGSYAY RANKL 155 evqlvesggglvqag217 NYVMG 279 wfrqapgker 341 AISTGGSWT 403 rftisrdntkntvylqmaslkpedt 465TMPATTYL 527 wgqgtqvtvss PMP7B2 gslrlscaaaggtfr efvt GYVDSVKD avyycaaPRSERQY DY RANKL 156 evqlvesggglvqag 218 RYVMG 280 wfrqapgker 342AISTGGTWT 404 rftisrdntkntvylqmaslkpedt 466 TTPTTSYL 528 wgqgtqvtvssPMP7A11 gsltlscaaagftfr efva GYVDSVKD avyncaa PRSERQY EY RANKL 157evqlvesggglvqag 219 NYVMG 281 wfrqapgker 343 AISTGGTWT 405rftisrdntkntvnlqmaslkped 467 TTPTTSYL 529 wgqgtqvtvss PMP7F1gslrlscaaagctfr efvt GYVDSVKD tavyycaa PRSERQY EY RANKL 158evqlvesggglvqag 220 NYVMG 282 wfrqapgker 344 AISTGGSWT 406rftisrdntkntvylqmvslkpedt 468 TTPATTYL 530 wgqgtqvtvss PMP7H5gslrlscaaaggtfr efva GYVDSVKD avyycaa PRSERQY DY RANKL 159evqlvesggglvqag 221 NYVMG 283 wfrqapgker 345 AISAGGSWT 407rftisrdntkntvylqmaslkpedt 469 TTPATTYL 531 wgqgtqvtvss PMP7E7gslrlscaaaggtfr efvt GYVDSVKD avyycaa PRSERQY DY RANKL 160evqlvesggglvqag 222 AYVMG 284 wfrqapgker 346 GISTGGTWT 408rftisrdntkntvylqmaslkpedt 470 TTPVTSYL 532 wgqgtqvtvss PMP7E2gslrlscaaagytfr efva GYVDSVKD avyycaa PRSERQY EH RANKL 161evqlvesggglvqsg 223 ARAYVMG 285 wfrqapgker 347 AISTGGTWT 409rftisrdntkntmylqmaslkpe 471 TTPSTSYL 533 wgqgtqvtvss PMP3H10gslrlscaaagytfr efva GYVDSVKD dtavyycaa PRSERQY EY RANKL 162evqlvesggglvqag 224 SYAMG 286 wfrqvpgker 348 AISTGSITIYG 410rftisrdnakntvylqmnslkped 472 GKREPYL 534 wgqgtqvtvss PMP7F9gslrlscvasrrtfs dfva DSVKG tavyycaa RQYTASN PYDY RANKL 163evqlvesggglvqag 225 SYAMG 287 wfrqvpgker 349 AITTGSITIYA 411rfaisrdnakntvylqmnslkpe 473 GNREPYL 535 wgqgtqvtvss PMP7E6gslrlscvaskrtfa dfva DSVKG dtavyycaa RQYTASN PYDY RANKL 164evqlvesggglvqvg 226 STYVMG 288 wfrqapgker 350 AVSWSSGNA 412rfatsrdtaknimylqmnslkpe 474 GRGYGLL 536 wgqgtqvtvss PMP4F4dslrlsceasgrsrf efva YYIDSAKG dtavytcaa SEYTQAP RYDY RANKL 165evqlvesggglvqvg 227 STYVMG 289 wfrqapgker 351 AISWSSGNA 413rfatsrdtaknimylqmnslkpe 475 GRGYGLL 537 wgqgtqvtvss PMP7B11dslrlsceasgrsrf efva YYIDSAKG dtavyscaa SEYTQAA RYDY RANKL 166evqlvesggglvqag 228 RSAMG 290 wfrqapgker 352 FITGSGGTTY 414rftisrdnaqnpvylqmnslkpe 476 YRRTYISS 538 wgqgtqvtvss PMP9H9gslrlscaasgrtfs efvg YGESVKG dtavyycgv TYSESSEY DY RANKL 167evqlvesggglvqag 229 RSAMG 291 wfrqapgker 353 FITGSGGTTY 415rftisrdnaqnpvylqmnslkpe 477 YRRTYISS 539 wgqgtqvtvss PMP9G3gslrlscaasgrtfs efvg YGESVKG dtavyycav TYNESSEY DY RANKL 168evqlvesggglvqag 230 RSAMG 292 wfrqapgker 354 FITGSGGTTY 416rftisrdnaqnpvylqmnslkpe 478 YRRTYISS 540 wgqgtqvtvss PMP9E3gslrlscaasgrtfs efvg YGESVKG dtavyycgv TYSESSEY DY RANKL 169evqlvesggglvqag 231 RSAMG 293 wfrqapgker 355 FITGSGGTTY 417rftisrdnaqnpvylqmnslkpe 479 YRRTYISIT 541 wgqgtqvtvss PMP7H9gslrlscaasgrtfs efvg YGESVKG dtavyycgv YSESSDY DY RANKL 170evqlvesggglvqag 232 ISAMG 294 wfrqapgker 356 FITGSGGTTY 418rftisrdnaqnpvylqmnslkpe 480 YRRTYISS 542 wgqgtqvtvss PMP4C3gslrlscaasgrtfs efvc YGESVKG dtavyycgv TYSESSEY DY RANKL 171evqlvesggglvqag 233 RSAMG 295 wfrqapgker 357 FITGSGGTTY 419rftisrdnaqnpvylqmnslkpe 481 YRRTYISS 543 wgqgtqvtvss PMP9G6gslrlscaasgrtfs efvg YGESVKG dtavyycgv TYSESSEY DY RANKL 172evqlvesggglvqag 234 RSAMG 296 wfrqapgker 358 FITGSGGTTY 420rftisrdnaqnpvylqmnslkpe 482 YRRTYISS 544 wgqgtqvtvss PMP7B12gslrlscaasgrtfs efvg YGESVKG dtavyycgv TYSESSEY DY RANKL 173evqlvesggglvqag 235 RSAMG 297 wfrqapgker 359 FITGSGGTTY 421rftisrdnaqnpvylqmnslkpe 483 YRRTYISS 545 wgqgtqvtvss PMP7G3gslrlscaasgrtfs efvg YGESVKG dtavyycav TYNESSEY DY RANKL 174evqlvesggglvqag 236 RSAMG 298 wfrqapgker 360 FITGSGGTTY 422rftisrdnaqnpvylqmnslkpe 484 YRRTYISS 546 wgqgtqvtvss PMP9C12gslrlscaasgrtfs efvg YGESVKG dtavyycgv TYSESSEY DY RANKL 175evqlvesggglvqag 237 MG 299 wfrqapgker 361 AISGSGSITN 423rftisrdyakttvflqmnslkpedt 485 YVRTPYYS 547 wgqgtqvtvss PMP1D8dslrlscaasgrift efva YADSVKG avyycaa SYYDSTKY EY RANKL 176evqlvesggglvqag 238 MG 300 wfrqapgker 362 FISGSGSVTN 424rftisrdhakntvflqmnslkped 486 YLRGPYYS 548 wgqgtqvtvss PMP1A2dslrlscaasgrtft efva YTDSVKG tavyycaa SFYDSTKY EY RANKL 177evqlvesggglvqag 239 MG 301 wfrrapgteref 363 SISGSGKITN 425rftisrdhaknavflqmdglkpe 487 YLRSPYYS 549 wgqgtqvtvss PMP1E5dslrlscaasgrtft va YADSVKG dtavyycaa SYYDSAKY EY RANKL 178evqlvesgggsvqa 240 MG 302 wfrqapgtere 364 AISGSGKITN 426rftisrdhamntvflqmdslkpe 488 YLRSPYYS 550 wgqgtqvtvss PMP2B8gdslrlscaasgrtft fva YADSVKG dtavyycaa SYYDSAKY EY RANKL 179evqlvesggglvqag 241 MG 303 wfrqapgtere 365 AISGSGKITN 427rftisrdhakntvflqmdslkped 489 YLRSPYYS 551 wgqgtqvtvss PMP2C5dslrlscaasgrtft fva YADSVKG tavyycaa SYYDSAKY EY RANKL 180evqlvesggglvqag 242 MG 304 wfrqapgtere 366 AISGSGKITN 428rftisrdhakntvflqmdslkped 490 YLRSPYYS 552 wgqgtqvtvss PMP2B4dslrlscaasgrtft fva YADSVKG tavyycaa SYYDSAKY EY RANKL 181evqlvesggglvqag 243 MG 305 wfrqapgtere 367 AISGSGKITN 429rftisrdhakntvflqmdslkped 491 YLRSPYYS 553 wgqgtqvtvss PMP2A5dslrlscaasgrtft fva YADSVKG tavyycaa SYYDSAKY EY RANKL 182evqlvesggglvqag 244 MG 306 wfrqapgtere 368 AISGSGKITN 430rftisrdhakntvflqmdslkped 492 YLRSPYYS 554 wgqgtqvtvss PMP2D7dslrlscaasgrtft fva YADSVKG tavyycaa SYYDSAKY EY RANKL 183evqlvesggglvqag 245 MG 307 wfrqapgtere 369 AISGSGKITN 431rftisrdhakntvflqmdslkped 493 YLRSPYYS 555 wgqgtqvtvss PMP2G4dslrlscaasgrtft fva YADSVKG tavyycaa SYYDSAKY EY RANKL 184emqlvesggglvqa 246 SYAMG 308 wfrqvpgker 370 AISTHSITVYA 432rftisrdnakntvylqmntlkped 494 GNREPYL 556 wgqgtqvtvss PMP7A8ggslrlscvaskrtfa dfva DSVKG tavyycaa RQYTASN PYDY RANKL 185evqlvesggglvqtg 247 SYAVG 309 wfrqvpgker 371 AISTGSVTIY 433rftisrdntkntvylqmnslkpedt 495 GNREPYL 557 wgqgtqvtvss PMP7A5gslrlscvasrrtfs dfva ADSVKG avyycaa RQYTASN PYDY RANKL 186 EVQLVESGGGL248 NYVMG 310 WFRQAPG 372 AISTGGSWT 434 RFTISRDNTKNTVYLH 496 TTPVTTYL558 WGQGTQVTV PMP7F8 VQAGGSLRLSC KEREFVT GYVDSVKD MASLKPEDTAVYYCAAPRSERQY SS AAAGGTFR DY RANKL 187 EVQLVESGGGL 249 RYVMG 311 WFRQAPG 373AISTGGTWT 435 RFTISRDNTKNTVYLQ 497 TTPTTSYL 559 WGQGTQVTV PMP7F6VQAGDSLRLSC KEREFVA GYVDSVKD MASLKPEDTAVYNCAA PRSERQY SS AAAGFTFR EY

Thus, in the NANOBODIES of the invention, at least one of the CDR1, CDR2and CDR3 sequences present is suitably chosen from the group consistingof the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1;or from the group of CDR1, CDR2 and CDR3 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% “sequence identity” (as definedherein) with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” (as defined herein) with at least one ofthe CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

In this context, by “suitably chosen” is meant that, as applicable, aCDR1 sequence is chosen from suitable CDR1 sequences (i.e. as definedherein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. asdefined herein), and a CDR3 sequence is chosen from suitable CDR3sequence (i.e. as defined herein), respectively. More in particular, theCDR sequences are preferably chosen such that the NANOBODIES of theinvention bind to RANK-L with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined herein.

In particular, in the NANOBODIES of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table A-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table A-1; and/or from thegroup consisting of the CDR3 sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the CDR3 sequences listed inTable A-1.

Preferably, in the NANOBODIES of the invention, at least two of theCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable A-1 or from the group consisting of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” with at least one of the CDR1, CDR2 andCDR3 sequences, respectively, listed in Table A-1.

In particular, in the NANOBODIES of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table A-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table A-1, respectively; andat least one of the CDR1 and CDR2 sequences present is suitably chosenfrom the group consisting of the CDR1 and CDR2 sequences, respectively,listed in Table A-1 or from the group of CDR1 and CDR2 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1 and CDR2 sequences, respectively,listed in Table A-1; and/or from the group consisting of the CDR1 andCDR2 sequences, respectively, that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR1 and CDR2 sequences,respectively, listed in Table A-1.

Most preferably, in the NANOBODIES of the invention, all three CDR1,CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable A-1 or from the group of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 amino acid difference(s) with at least one of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table A-1.

Even more preferably, in the NANOBODIES of the invention, at least oneof the CDR1, CDR2 and CDR3 sequences present is suitably chosen from thegroup consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table A-1. Preferably, in this aspect, at least one orpreferably both of the other two CDR sequences present are suitablychosen from CDR sequences that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with at least one of the corresponding CDR sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR sequences that have 3, 2 or only 1 amino acid difference(s) withat least one of the corresponding sequences, respectively, listed inTable A-1.

In particular, in the NANOBODIES of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 listed in Table A-1. Preferably, in this aspect, at least one andpreferably both of the CDR1 and CDR2 sequences present are suitablychosen from the groups of CDR1 and CDR2 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with the CDR1and CDR2 sequences, respectively, listed in Table A-1; and/or from thegroup consisting of the CDR1 and CDR2 sequences, respectively, that have3, 2 or only 1 amino acid difference(s) with at least one of the CDR1and CDR2 sequences, respectively, listed in Table A-1.

Even more preferably, in the NANOBODIES of the invention, at least twoof the CDR1, CDR2 and CDR3 sequences present are suitably chosen fromthe group consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table A-1. Preferably, in this aspect, the remaining CDRsequence present is suitably chosen from the group of CDR sequences thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with at leastone of the corresponding CDR sequences listed in Table A-1; and/or fromthe group consisting of CDR sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the corresponding sequenceslisted in Table A-1.

In particular, in the NANOBODIES of the invention, at least the CDR3sequence is suitably chosen from the group consisting of the CDR3sequences listed in Table A-1, and either the CDR1 sequence or the CDR2sequence is suitably chosen from the group consisting of the CDR1 andCDR2 sequences, respectively, listed in Table A-1. Preferably, in thisaspect, the remaining CDR sequence present is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the corresponding CDR sequences listed inTable A-1; and/or from the group consisting of CDR sequences that have3, 2 or only 1 amino acid difference(s) with the corresponding CDRsequences listed in Table A-1.

Even more preferably, in the NANOBODIES of the invention, all threeCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable A-1.

Also, generally, the combinations of CDR's listed in Table A-1 (i.e.those mentioned on the same line in Table A-1) are preferred. Thus, itis generally preferred that, when a CDR in a NANOBODY of the inventionis a CDR sequence mentioned in Table A-1 or is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with a CDR sequence listed in Table A-1; and/or from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with a CDR sequence listed in Table A-1, that at least oneand preferably both of the other CDR's are suitably chosen from the CDRsequences that belong to the same combination in Table A-1 (i.e.mentioned on the same line in Table A-1) or are suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with the CDR sequence(s) belonging to the same combinationand/or from the group consisting of CDR sequences that have 3, 2 or only1 amino acid difference(s) with the CDR sequence(s) belonging to thesame combination. The other preferences indicated in the aboveparagraphs also apply to the combinations of CDR's mentioned in TableA-1.

Thus, by means of non-limiting examples, a NANOBODY of the invention canfor example comprise a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table A-1, a CDR2sequence that has 3, 2 or 1 amino acid difference with one of the CDR2sequences mentioned in Table A-1 (but belonging to a differentcombination), and a CDR3 sequence.

Some preferred NANOBODIES of the invention may for example comprise: (1)a CDR1 sequence that has more than 80% sequence identity with one of theCDR1 sequences mentioned in Table A-1; a CDR2 sequence that has 3, 2 or1 amino acid difference with one of the CDR2 sequences mentioned inTable A-1 (but belonging to a different combination); and a CDR3sequence that has more than 80% sequence identity with one of the CDR3sequences mentioned in Table A-1 (but belonging to a differentcombination); or (2) a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table A-1; a CDR2sequence, and one of the CDR3 sequences listed in Table A-1; or (3) aCDR1 sequence; a CDR2 sequence that has more than 80% sequence identitywith one of the CDR2 sequence listed in Table A-1; and a CDR3 sequencethat has 3, 2 or 1 amino acid differences with the CDR3 sequencementioned in Table A-1 that belongs to the same combination as the CDR2sequence.

Some particularly preferred NANOBODIES of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table A-1; a CDR2 sequencethat has 3, 2 or 1 amino acid difference with the CDR2 sequencementioned in Table A-1 that belongs to the same combination; and a CDR3sequence that has more than 80% sequence identity with the CDR3 sequencementioned in Table A-1 that belongs to the same combination; (2) a CDR1sequence; a CDR 2 listed in Table A-1 and a CDR3 sequence listed inTable A-1 (in which the CDR2 sequence and CDR3 sequence may belong todifferent combinations).

Some even more preferred NANOBODIES of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table A-1; the CDR2 sequencelisted in Table A-1 that belongs to the same combination; and a CDR3sequence mentioned in Table A-1 that belongs to a different combination;or (2) a CDR1 sequence mentioned in Table A-1; a CDR2 sequence that has3, 2 or 1 amino acid differences with the CDR2 sequence mentioned inTable A-1 that belongs to the same combination; and a CDR3 sequence thathas more than 80% sequence identity with the CDR3 sequence listed inTable A-1 that belongs to the same or a different combination.

Particularly preferred NANOBODIES of the invention may for examplecomprise a CDR1 sequence mentioned in Table A-1, a CDR2 sequence thathas more than 80% sequence identity with the CDR2 sequence mentioned inTable A-1 that belongs to the same combination; and the CDR3 sequencementioned in Table A-1 that belongs to the same combination.

In the most preferred NANOBODIES of the invention, the CDR1, CDR2 andCDR3 sequences present are suitably chosen from one of the combinationsof CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

According to another preferred, but non-limiting aspect of the invention(a) CDR1 has a length of between 1 and 12 amino acid residues, andusually between 2 and 9 amino acid residues, such as 5, 6 or 7 aminoacid residues; and/or (b) CDR2 has a length of between 13 and 24 aminoacid residues, and usually between 15 and 21 amino acid residues, suchas 16 and 17 amino acid residues; and/or (c) CDR3 has a length ofbetween 2 and 35 amino acid residues, and usually between 3 and 30 aminoacid residues, such as between 6 and 23 amino acid residues.

In another preferred, but non-limiting aspect, the invention relates toa NANOBODY in which the CDR sequences (as defined herein) have more than80%, preferably more than 90%, more preferably more than 95%, such as99% or more sequence identity (as defined herein) with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 560-621.

Generally, NANOBODIES with the above CDR sequences may be as furtherdescribed herein, and preferably have framework sequences that are alsoas further described herein. Thus, for example and as mentioned herein,such NANOBODIES may be naturally occurring NANOBODIES (from any suitablespecies), naturally occurring V_(HH) sequences (i.e. from a suitablespecies of Camelid) or synthetic or semi-synthetic amino acid sequencesor NANOBODIES, including but not limited to partially humanizedNANOBODIES or V_(HH) sequences, fully humanized NANOBODIES or V_(HH)sequences, camelized heavy chain variable domain sequences, as well asNANOBODIES that have been obtained by the techniques mentioned herein.

Thus, in one specific, but non-limiting aspect, the invention relates toa humanized NANOBODY, which consists of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), in which CDR1 to CDR3 are as defined herein and in whichsaid humanized NANOBODY comprises at least one humanizing substitution(as defined herein), and in particular at least one humanizingsubstitution in at least one of its framework sequences (as definedherein).

In another preferred, but non-limiting aspect, the invention relates toa NANOBODY in which the CDR sequences have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 560-621.This degree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said NANOBODY and one or more of the sequences of SEQ IDNO's: 560-621, in which the amino acid residues that form the frameworkregions are disregarded. Such NANOBODIES can be as further describedherein.

In another preferred, but non-limiting aspect, the invention relates toa NANOBODY with an amino acid sequence that is chosen from the groupconsisting of SEQ ID NO's: 560-621 or from the group consisting of fromamino acid sequences that have more than 80%, preferably more than 90%,more preferably more than 95%, such as 99% or more sequence identity (asdefined herein) with at least one of the amino acid sequences of SEQ IDNO's: 560-621.

Another preferred, but non-limiting aspect of the invention relates tohumanized variants of the NANOBODIES of SEQ ID NO's: 560-621, thatcomprise, compared to the corresponding native V_(HH) sequence, at leastone humanizing substitution (as defined herein), and in particular atleast one humanizing substitution in at least one of its frameworksequences (as defined herein). Some preferred, but non-limiting examplesof such humanized variants are the humanized NANOBODIES of SEQ ID NO's:730-757 and 765. Thus, the invention also relates to a humanizedNANOBODY with an amino acid sequence that is chosen from the groupconsisting of SEQ ID NO's: 730-757 and 765 or from the group consistingof from amino acid sequences that have more than 80%, preferably morethan 90%, more preferably more than 95%, such as 99% or more sequenceidentity (as defined herein) with at least one of the amino acidsequences of SEQ ID NO's: 730-757 and 765 (in which amino acid sequencesthat are chosen from the latter group of amino acid sequences maycontain a greater number or a smaller number of humanizing substitutionscompared to the corresponding sequence of SEQ ID NO's: 730-757 and 765,as long as they retain at least one of the humanizing substitutionspresent in the corresponding sequence of SEQ ID NO's: 730-757 and 765).

The polypeptides of the invention comprise or essentially consist of atleast one NANOBODY of the invention. Some preferred, but non-limitingexamples of polypeptides of the invention are given in SEQ ID NO's:622-729, 759-762 and 766-789.

It will be clear to the skilled person that the NANOBODIES that arementioned herein as “preferred” (or “more preferred”, “even morepreferred”, etc.) are also preferred (or more preferred, or even morepreferred, etc.) for use in the polypeptides described herein. Thus,polypeptides that comprise or essentially consist of one or more“preferred” NANOBODIES of the invention will generally be preferred, andpolypeptides that comprise or essentially consist of one or more “morepreferred” NANOBODIES of the invention will generally be more preferred,etc.

Generally, proteins or polypeptides that comprise or essentially consistof a single NANOBODY (such as a single NANOBODY of the invention) willbe referred to herein as “monovalent” proteins or polypeptides or as“monovalent constructs”. Proteins and polypeptides that comprise oressentially consist of two or more NANOBODIES (such as at least twoNANOBODIES of the invention or at least one NANOBODY of the inventionand at least one other NANOBODY) will be referred to herein as“multivalent” proteins or polypeptides or as “multivalent constructs”,and these may provide certain advantages compared to the correspondingmonovalent NANOBODIES of the invention. Some non-limiting examples ofsuch multivalent constructs will become clear from the furtherdescription herein.

According to one specific, but non-limiting aspect, a polypeptide of theinvention comprises or essentially consists of at least two NANOBODIESof the invention, such as two or three NANOBODIES of the invention. Asfurther described herein, such multivalent constructs can providecertain advantages compared to a protein or polypeptide comprising oressentially consisting of a single NANOBODY of the invention, such as amuch improved avidity for RANK-L. Such multivalent constructs will beclear to the skilled person based on the disclosure herein; somepreferred, but non-limiting examples of such multivalent NANOBODYconstructs are the constructs of SEQ ID NO's: 622-693, 761-762 and766-773 (bivalent) and SEQ ID NO's: 694-729 and 759-760 (trivalent).

According to another specific, but non-limiting aspect, a polypeptide ofthe invention comprises or essentially consists of at least one NANOBODYof the invention and at least one other binding unit (i.e. directedagainst another epitope, antigen, target, protein or polypeptide), whichis preferably also a NANOBODY. Such proteins or polypeptides are alsoreferred to herein as “multispecific” proteins or polypeptides or as‘multispecific constructs”, and these may provide certain advantagescompared to the corresponding monovalent NANOBODIES of the invention (aswill become clear from the further discussion herein of some preferred,but-nonlimiting multispecific constructs). Such multispecific constructswill be clear to the skilled person based on the disclosure herein; somepreferred, but non-limiting examples of such multispecific NANOBODYconstructs are the constructs of SEQ ID NO's: 694-729 and 759-790.

According to yet another specific, but non-limiting aspect, apolypeptide of the invention comprises or essentially consists of atleast one NANOBODY of the invention, optionally one or more furtherNANOBODIES, and at least one other amino acid sequence (such as aprotein or polypeptide) that confers at least one desired property tothe NANOBODY of the invention and/or to the resulting fusion protein.Again, such fusion proteins may provide certain advantages compared tothe corresponding monovalent NANOBODIES of the invention. Somenon-limiting examples of such amino acid sequences and of such fusionconstructs will become clear from the further description herein.

It is also possible to combine two or more of the above aspects, forexample to provide a trivalent bispecific construct comprising twoNANOBODIES of the invention and one other NANOBODY, and optionally oneor more other amino acid sequences. Further non-limiting examples ofsuch constructs, as well as some constructs that are particularlypreferred within the context of the present invention, will become clearfrom the further description herein.

In the above constructs, the one or more NANOBODIES and/or other aminoacid sequences may be directly linked to each other and/or suitablylinked to each other via one or more linker sequences. Some suitable butnon-limiting examples of such linkers will become clear from the furtherdescription herein.

In one specific aspect of the invention, a NANOBODY of the invention ora compound, construct or polypeptide of the invention comprising atleast one NANOBODY of the invention may have an increased half-life,compared to the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such NANOBODIES, compounds andpolypeptides will become clear to the skilled person based on thefurther disclosure herein, and for example comprise NANOBODIES sequencesor polypeptides of the invention that have been chemically modified toincrease the half-life thereof (for example, by means of pegylation);amino acid sequences of the invention that comprise at least oneadditional binding site for binding to a serum protein (such as serumalbumin); or polypeptides of the invention that comprise at least oneNANOBODY of the invention that is linked to at least one moiety (and inparticular at least one amino acid sequence) that increases thehalf-life of the NANOBODY of the invention. Examples of polypeptides ofthe invention that comprise such half-life extending moieties or aminoacid sequences will become clear to the skilled person based on thefurther disclosure herein; and for example include, without limitation,polypeptides in which the one or more NANOBODIES of the invention aresuitable linked to one or more serum proteins or fragments thereof (suchas serum albumin or suitable fragments thereof) or to one or morebinding units that can bind to serum proteins (such as, for example,NANOBODIES or (single) domain antibodies that can bind to serum proteinssuch as serum albumin, serum immunoglobulins such as IgG, ortransferrin); polypeptides in which a NANOBODY of the invention islinked to an Fc portion (such as a human Fc) or a suitable part orfragment thereof (the invention, for examples envisages polypeptides inwhich the NANOBODIES are suitably linked to an Fc portion by linkers indifferent sizes to allow intra and/or intermolecular binding of RANK-L);or polypeptides in which the one or more NANOBODIES of the invention aresuitable linked to one or more small proteins or peptides that can bindto serum proteins (such as, without limitation, the proteins andpeptides described in WO 91/01743, WO 01/45746, WO 02/076489 and to theUS provisional application of Ablynx N.V. entitled “Peptides capable ofbinding to serum proteins” of Ablynx N.V. filed on Dec. 5, 2006 (seealso PCT/EP2007/063348).

Again, as will be clear to the skilled person, such NANOBODIES,compounds, constructs or polypeptides may contain one or more additionalgroups, residues, moieties or binding units, such as one or more furtheramino acid sequences and in particular one or more additional NANOBODIES(i.e. not directed against RANK-L), so as to provide a tri- ofmultispecific NANOBODY construct.

Generally, the NANOBODIES of the invention (or compounds, constructs orpolypeptides comprising the same) with increased half-life preferablyhave a half-life that is at least 1.5 times, preferably at least 2times, such as at least 5 times, for example at least 10 times or morethan 20 times, greater than the half-life of the corresponding aminoacid sequence of the invention per se. For example, the NANOBODIES,compounds, constructs or polypeptides of the invention with increasedhalf-life may have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchNANOBODIES, compound, constructs or polypeptides of the inventionexhibit a serum half-life in human of at least about 12 hours,preferably at least 24 hours, more preferably at least 48 hours, evenmore preferably at least 72 hours or more. For example, compounds orpolypeptides of the invention may have a half-life of at least 5 days(such as about 5 to 10 days), preferably at least 9 days (such as about9 to 14 days), more preferably at least about 10 days (such as about 10to 15 days), or at least about 11 days (such as about 11 to 16 days),more preferably at least about 12 days (such as about 12 to 18 days ormore), or more than 14 days (such as about 14 to 19 days).

In another one aspect of the invention, a polypeptide of the inventioncomprises one or more (such as two or preferably one) NANOBODIES of theinvention linked (optionally via one or more suitable linker sequences)to one or more (such as two and preferably one) amino acid sequencesthat allow the resulting polypeptide of the invention to cross the bloodbrain barrier. In particular, said one or more amino acid sequences thatallow the resulting polypeptides of the invention to cross the bloodbrain barrier may be one or more (such as two and preferably one)NANOBODIES, such as the NANOBODIES described in WO 02/057445, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

In particular, polypeptides comprising one or more NANOBODIES of theinvention are preferably such that they:

-   -   bind to RANK-L with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to RANK-L with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more        preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between        10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to RANK-L with a k_(off) rate between 1 s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶        s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a polypeptide that contains only one amino acid sequence ofthe invention is preferably such that it will bind to RANK-L with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM, such as less than 500 pM. In this respect, it will beclear to the skilled person that a polypeptide that contains two or moreNANOBODIES of the invention may bind to RANK-L with an increasedavidity, compared to a polypeptide that contains only one amino acidsequence of the invention.

Some preferred IC₅₀ values for binding of the amino acid sequences orpolypeptides of the invention to RANK-L will become clear from thefurther description and examples herein.

Other polypeptides according to this preferred aspect of the inventionmay for example be chosen from the group consisting of amino acidsequences that have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more “sequence identity” (asdefined herein) with one or more of the amino acid sequences of SEQ IDNO's: 622-729, 759-762 and 766-773, in which the NANOBODIES comprisedwithin said amino acid sequences are preferably as further definedherein.

Another aspect of this invention relates to a nucleic acid that encodesan amino acid sequence of the invention (such as a NANOBODY of theinvention) or a polypeptide of the invention comprising the same. Again,as generally described herein for the nucleic acids of the invention,such a nucleic acid may be in the form of a genetic construct, asdefined herein.

In another aspect, the invention relates to host or host cell thatexpresses or that is capable of expressing an amino acid sequence (suchas a NANOBODY) of the invention and/or a polypeptide of the inventioncomprising the same; and/or that contains a nucleic acid of theinvention. Some preferred but non-limiting examples of such hosts orhost cells will become clear from the further description herein.

Another aspect of the invention relates to a product or compositioncontaining or comprising at least one amino acid sequence of theinvention, at least one polypeptide of the invention and/or at least onenucleic acid of the invention, and optionally one or more furthercomponents of such compositions known per se, i.e. depending on theintended use of the composition. Such a product or composition may forexample be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or compositions will become clear from the furtherdescription herein.

The invention further relates to methods for preparing or generating theamino acid sequences, compounds, constructs, polypeptides, nucleicacids, host cells, products and compositions described herein. Somepreferred but non-limiting examples of such methods will become clearfrom the further description herein.

The invention further relates to applications and uses of the amino acidsequences, compounds, constructs, polypeptides, nucleic acids, hostcells, products and compositions described herein, as well as to methodsfor the prevention and/or treatment for diseases and disordersassociated with RANK-L. Some preferred but non-limiting applications anduses will become clear from the further description herein.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description hereinbelow.

Generally, it should be noted that the term NANOBODY as used herein inits broadest sense is not limited to a specific biological source or toa specific method of preparation. For example, as will be discussed inmore detail below, the NANOBODIES of the invention can generally beobtained: (1) by isolating the V_(HH) domain of a naturally occurringheavy chain antibody; (2) by expression of a nucleotide sequenceencoding a naturally occurring V_(HH) domain; (3) by “humanization” (asdescribed herein) of a naturally occurring V_(HH) domain or byexpression of a nucleic acid encoding a such humanized V_(HH) domain;(4) by “camelization” (as described herein) of a naturally occurringV_(H) domain from any animal species, and in particular a from speciesof mammal, such as from a human being, or by expression of a nucleicacid encoding such a camelized V_(H) domain; (5) by “camelisation” of a“domain antibody” or “Dab” as described by Ward et al (supra), or byexpression of a nucleic acid encoding such a camelized V_(H) domain; (6)by using synthetic or semi-synthetic techniques for preparing proteins,polypeptides or other amino acid sequences known per se; (7) bypreparing a nucleic acid encoding a NANOBODY using techniques fornucleic acid synthesis known per se, followed by expression of thenucleic acid thus obtained; and/or (8) by any combination of one or moreof the foregoing. Suitable methods and techniques for performing theforegoing will be clear to the skilled person based on the disclosureherein and for example include the methods and techniques described inmore detail herein.

One preferred class of NANOBODIES corresponds to the V_(HH) domains ofnaturally occurring heavy chain antibodies directed against RANK-L. Asfurther described herein, such V_(HH) sequences can generally begenerated or obtained by suitably immunizing a species of Camelid withRANK-L (i.e. so as to raise an immune response and/or heavy chainantibodies directed against RANK-L), by obtaining a suitable biologicalsample from said Camelid (such as a blood sample, serum sample or sampleof B-cells), and by generating V_(HH) sequences directed against RANK-L,starting from said sample, using any suitable technique known per se.Such techniques will be clear to the skilled person and/or are furtherdescribed herein.

Alternatively, such naturally occurring V_(HH) domains against RANK-L,can be obtained from naïve libraries of Camelid V_(HH) sequences, forexample by screening such a library using RANK-L, or at least one part,fragment, antigenic determinant or epitope thereof using one or morescreening techniques known per se. Such libraries and techniques are forexample described in WO 99/37681, WO 01/90190, WO 03/025020 and WO03/035694. Alternatively, improved synthetic or semi-synthetic librariesderived from naïve V_(HH) libraries may be used, such as V_(HH)libraries obtained from naïve V_(HH) libraries by techniques such asrandom mutagenesis and/or CDR shuffling, as for example described in WO00/43507.

Thus, in another aspect, the invention relates to a method forgenerating NANOBODIES, that are directed against RANK-L. In one aspect,said method at least comprises the steps of:

-   a) providing a set, collection or library of NANOBODY sequences; and-   b) screening said set, collection or library of NANOBODY sequences    for NANOBODY sequences that can bind to and/or have affinity for    RANK-L;    and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for RANK-L.

In such a method, the set, collection or library of NANOBODY sequencesmay be a naïve set, collection or library of NANOBODY sequences; asynthetic or semi-synthetic set, collection or library of NANOBODYsequences; and/or a set, collection or library of NANOBODY sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofNANOBODY sequences may be an immune set, collection or library ofNANOBODY sequences, and in particular an immune set, collection orlibrary of V_(HH) sequences, that have been derived from a species ofCamelid that has been suitably immunized with RANK-L or with a suitableantigenic determinant based thereon or derived therefrom, such as anantigenic part, fragment, region, domain, loop or other epitope thereof.In one particular aspect, said antigenic determinant may be anextracellular part, region, domain, loop or other extracellularepitope(s).

In the above methods, the set, collection or library of NANOBODY orV_(HH) sequences may be displayed on a phage, phagemid, ribosome orsuitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of) NANOBODYsequences will be clear to the person skilled in the art, for example onthe basis of the further disclosure herein. Reference is also made to WO03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23,9, 1105-1116 (2005).

In another aspect, the method for generating NANOBODY sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells derived from a species    of Camelid that express immunoglobulin sequences;-   b) screening said collection or sample of cells for (i) cells that    express an immunoglobulin sequence that can bind to and/or have    affinity for RANK-L; and (ii) cells that express heavy chain    antibodies, in which substeps (i) and (ii) can be performed    essentially as a single screening step or in any suitable order as    two separate screening steps, so as to provide at least one cell    that expresses a heavy chain antibody that can bind to and/or has    affinity for RANK-L;    and-   c) either (i) isolating from said cell the V_(HH) sequence present    in said heavy chain antibody; or (ii) isolating from said cell a    nucleic acid sequence that encodes the V_(HH) sequence present in    said heavy chain antibody, followed by expressing said V_(HH)    domain.

In the method according to this aspect, the collection or sample ofcells may for example be a collection or sample of B-cells. Also, inthis method, the sample of cells may be derived from a Camelid that hasbeen suitably immunized with RANK-L or a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820. Particular reference is made to the so-called“Nanoclone™” technique described in International application WO06/079372 by Ablynx N.V.

In another aspect, the method for generating an amino acid sequencedirected against RANK-L may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding heavy chain antibodies or NANOBODY sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode a heavy chain    antibody or a NANOBODY sequence that can bind to and/or has affinity    for RANK-L;    and-   c) isolating said nucleic acid sequence, followed by expressing the    V_(HH) sequence present in said heavy chain antibody or by    expressing said NANOBODY sequence, respectively.

In such a method, the set, collection or library of nucleic acidsequences encoding heavy chain antibodies or NANOBODY sequences may forexample be a set, collection or library of nucleic acid sequencesencoding a naïve set, collection or library of heavy chain antibodies orV_(HH) sequences; a set, collection or library of nucleic acid sequencesencoding a synthetic or semi-synthetic set, collection or library ofNANOBODY sequences; and/or a set, collection or library of nucleic acidsequences encoding a set, collection or library of NANOBODY sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofnucleic acid sequences encoding heavy chain antibodies or V_(HH)sequences derived from a Camelid that has been suitably immunized withRANK-L or with a suitable antigenic determinant based thereon or derivedtherefrom, such as an antigenic part, fragment, region, domain, loop orother epitope thereof. In one particular aspect, said antigenicdeterminant may be an extracellular part, region, domain, loop or otherextracellular epitope(s).

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, for example on the basis of the further disclosure herein.Reference is also made to WO 03/054016 and to the review by Hoogenboomin Nature Biotechnology, 23, 9, 1105-1116 (2005).

As will be clear to the skilled person, the screening step of themethods described herein can also be performed as a selection step.Accordingly the term “screening” as used in the present description cancomprise selection, screening or any suitable combination of selectionand/or screening techniques. Also, when a set, collection or library ofsequences is used, it may contain any suitable number of sequences, suchas 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 10⁴, 10⁵, 10⁶, 10⁷,10⁸ or more sequences.

Also, one or more or all of the sequences in the above set, collectionor library of amino acid sequences may be obtained or defined byrational, or semi-empirical approaches such as computer modellingtechniques or biostatics or datamining techniques.

Furthermore, such a set, collection or library can comprise one, two ormore sequences that are variants from one another (e.g. with designedpoint mutations or with randomized positions), compromise multiplesequences derived from a diverse set of naturally diversified sequences(e.g. an immune library)), or any other source of diverse sequences (asdescribed for example in Hoogenboom et al, Nat Biotechnol 23:1105, 2005and Binz et al, Nat Biotechnol 2005, 23:1247). Such set, collection orlibrary of sequences can be displayed on the surface of a phageparticle, a ribosome, a bacterium, a yeast cell, a mammalian cell, andlinked to the nucleotide sequence encoding the amino acid sequencewithin these carriers. This makes such set, collection or libraryamenable to selection procedures to isolate the desired amino acidsequences of the invention. More generally, when a sequence is displayedon a suitable host or host cell, it is also possible (and customary) tofirst isolate from said host or host cell a nucleotide sequence thatencodes the desired sequence, and then to obtain the desired sequence bysuitably expressing said nucleotide sequence in a suitable hostorganism. Again, this can be performed in any suitable manner known perse, as will be clear to the skilled person.

Yet another technique for obtaining V_(HH) sequences or NANOBODYsequences directed against RANK-L involves suitably immunizing atransgenic mammal that is capable of expressing heavy chain antibodies(i.e. so as to raise an immune response and/or heavy chain antibodiesdirected against RANK-L), obtaining a suitable biological sample fromsaid transgenic mammal that contains (nucleic acid sequences encoding)said V_(HH) sequences or NANOBODY sequences (such as a blood sample,serum sample or sample of B-cells), and then generating V_(HH) sequencesdirected against RANK-L, starting from said sample, using any suitabletechnique known per se (such as any of the methods described herein or ahybridoma technique). For example, for this purpose, the heavy chainantibody-expressing mice and the further methods and techniquesdescribed in WO 02/085945, WO 04/049794 and WO 06/008548 and Janssens etal., Proc. Natl. Acad. Sci. USA. 2006 Oct. 10; 103(41):15130-5 can beused. For example, such heavy chain antibody expressing mice can expressheavy chain antibodies with any suitable (single) variable domain, suchas (single) variable domains from natural sources (e.g. human (single)variable domains, Camelid (single) variable domains or shark (single)variable domains), as well as for example synthetic or semi-synthetic(single) variable domains.

The invention also relates to the V_(HH) sequences or NANOBODY sequencesthat are obtained by the above methods, or alternatively by a methodthat comprises the one of the above methods and in addition at least thesteps of determining the nucleotide sequence or amino acid sequence ofsaid V_(HH) sequence or NANOBODY sequence; and of expressing orsynthesizing said V_(HH) sequence or NANOBODY sequence in a manner knownper se, such as by expression in a suitable host cell or host organismor by chemical synthesis.

As mentioned herein, a particularly preferred class of NANOBODIES of theinvention comprises NANOBODIES with an amino acid sequence thatcorresponds to the amino acid sequence of a naturally occurring V_(HH)domain, but that has been “humanized”, i.e. by replacing one or moreamino acid residues in the amino acid sequence of said naturallyoccurring V_(HH) sequence (and in particular in the framework sequences)by one or more of the amino acid residues that occur at thecorresponding position(s) in a V_(H) domain from a conventional 4-chainantibody from a human being (e.g. indicated above). This can beperformed in a manner known per se, which will be clear to the skilledperson, for example on the basis of the further description herein andthe prior art on humanization referred to herein. Again, it should benoted that such humanized NANOBODIES of the invention can be obtained inany suitable manner known per se (i.e. as indicated under points (1)-(8)above) and thus are not strictly limited to polypeptides that have beenobtained using a polypeptide that comprises a naturally occurring V_(HH)domain as a starting material.

Another particularly preferred class of NANOBODIES of the inventioncomprises NANOBODIES with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring V_(H) domain, but that hasbeen “camelized”, i.e. by replacing one or more amino acid residues inthe amino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody. This can be performed in a manner known per se,which will be clear to the skilled person, for example on the basis ofthe further description herein. Such “camelizing” substitutions arepreferably inserted at amino acid positions that form and/or are presentat the V_(H)—V_(L) interface, and/or at the so-called Camelidae hallmarkresidues, as defined herein (see for example WO 94/04678 and Davies andRiechmann (1994 and 1996), supra). Preferably, the V_(H) sequence thatis used as a starting material or starting point for generating ordesigning the camelized NANOBODY is preferably a V_(H) sequence from amammal, more preferably the V_(H) sequence of a human being, such as aV_(H)3 sequence. However, it should be noted that such camelizedNANOBODIES of the invention can be obtained in any suitable manner knownper se (i.e. as indicated under points (1)-(8) above) and thus are notstrictly limited to polypeptides that have been obtained using apolypeptide that comprises a naturally occurring V_(H) domain as astarting material.

For example, again as further described herein, both “humanization” and“camelization” can be performed by providing a nucleotide sequence thatencodes a naturally occurring V_(HH) domain or V_(H) domain,respectively, and then changing, in a manner known per se, one or morecodons in said nucleotide sequence in such a way that the new nucleotidesequence encodes a “humanized” or “camelized” NANOBODY of the invention,respectively. This nucleic acid can then be expressed in a manner knownper se, so as to provide the desired NANOBODY of the invention.Alternatively, based on the amino acid sequence of a naturally occurringV_(HH) domain or V_(H) domain, respectively, the amino acid sequence ofthe desired humanized or camelized NANOBODY of the invention,respectively, can be designed and then synthesized de novo usingtechniques for peptide synthesis known per se. Also, based on the aminoacid sequence or nucleotide sequence of a naturally occurring V_(HH)domain or V_(H) domain, respectively, a nucleotide sequence encoding thedesired humanized or camelized NANOBODY of the invention, respectively,can be designed and then synthesized de novo using techniques fornucleic acid synthesis known per se, after which the nucleic acid thusobtained can be expressed in a manner known per se, so as to provide thedesired NANOBODY of the invention.

Other suitable methods and techniques for obtaining the NANOBODIES ofthe invention and/or nucleic acids encoding the same, starting fromnaturally occurring V_(H) sequences or preferably V_(HH) sequences, willbe clear from the skilled person, and may for example comprise combiningone or more parts of one or more naturally occurring V_(H) sequences(such as one or more FR sequences and/or CDR sequences), one or moreparts of one or more naturally occurring V_(HH) sequences (such as oneor more FR sequences or CDR sequences), and/or one or more synthetic orsemi-synthetic sequences, in a suitable manner, so as to provide aNANOBODY of the invention or a nucleotide sequence or nucleic acidencoding the same (which may then be suitably expressed). Nucleotidesequences encoding framework sequences of V_(HH) sequences or NANOBODIESwill be clear to the skilled person based on the disclosure hereinand/or the further prior art cited herein (and/or may alternatively beobtained by PCR starting from the nucleotide sequences obtained usingthe methods described herein) and may be suitably combined withnucleotide sequences that encode the desired CDR's (for example, by PCRassembly using overlapping primers), so as to provide a nucleic acidencoding a NANOBODY of the invention.

As mentioned herein, NANOBODIES may in particular be characterized bythe presence of one or more “Hallmark residues” (as described herein) inone or more of the framework sequences.

Thus, according to one preferred, but non-limiting aspect of theinvention, a NANOBODY in its broadest sense can be generally defined asa polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 45    according to the Kabat numbering is a charged amino acid (as defined    herein) or a cysteine residue, and position 44 is preferably an E;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, in a first preferred, but non-limiting aspect, a NANOBODY of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q; and/or in which:

-   b) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid or a cysteine and the amino acid    residue at position 44 according to the Kabat numbering is    preferably E;    and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, a NANOBODY in its broadest sense can be generally definedas a polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 44    according to the Kabat numbering is E and in which the amino acid    residue at position 45 according to the Kabat numbering is an R;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, according to a preferred, but non-limiting aspect, a NANOBODY ofthe invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q; and/or in which:

-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, a NANOBODY against RANK-L according to the invention mayhave the structure:

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:

-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, according to one preferred, but non-limiting aspect ofthe invention, a NANOBODY can generally be defined as a polypeptidecomprising an amino acid sequence that is comprised of four frameworkregions/sequences interrupted by three complementarity determiningregions/sequences, in which;

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q; and-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R; and-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q;    or in which:-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q; and-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R; and-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    or in which:-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q; and-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R; and-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S; and-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

Thus, in another preferred, but non-limiting aspect, a NANOBODY of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q;    and in which:

-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R;    and in which:

-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;    and in which

-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a NANOBODY of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q;    and in which:

-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R;    and in which:

-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;    and in which:

-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a NANOBODY of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q;    and in which:

-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R;    and in which:

-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S;    and in which:

-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

Two particularly preferred, but non-limiting groups of the NANOBODIES ofthe invention are those according to a) above; according to (a-1) to(a-4) above; according to b) above; according to (b-1) to (b-4) above;according to (c) above; and/or according to (c-1) to (c-4) above, inwhich either:

-   -   i) the amino acid residues at positions 44-47 according to the        Kabat numbering form the sequence GLEW (SEQ ID NO: 817) (or a        GLEW-like sequence as described herein) and the amino acid        residue at position 108 is Q;        or in which:    -   ii) the amino acid residues at positions 43-46 according to the        Kabat numbering form the sequence KERE (SEQ ID NO: 818) or KQRE        (SEQ ID NO: 819) (or a KERE-like sequence as described) and the        amino acid residue at position 108 is Q or L, and is preferably        Q.

Thus, in another preferred, but non-limiting aspect, a NANOBODY of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (SEQ ID NO: 817) (or a GLEW-like    sequence as defined herein) and the amino acid residue at position    108 is Q;    and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a NANOBODY of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) the amino acid residues at positions 43-46 according to the Kabat    numbering form the sequence KERE (SEQ ID NO: 818) or KQRE (SEQ ID    NO: 819) (or a KERE-like sequence) and the amino acid residue at    position 108 is Q or L, and is preferably Q;    and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the NANOBODIES of the invention in which the amino acid residues atpositions 43-46 according to the Kabat numbering form the sequence KERE(SEQ ID NO: 818) or KQRE (SEQ ID NO: 819), the amino acid residue atposition 37 is most preferably F. In the NANOBODIES of the invention inwhich the amino acid residues at positions 44-47 according to the Kabatnumbering form the sequence GLEW (SEQ ID NO: 817), the amino acidresidue at position 37 is chosen from the group consisting of Y, H, I,L, V or F, and is most preferably V.

Thus, without being limited hereto in any way, on the basis of the aminoacid residues present on the positions mentioned above, the NANOBODIESof the invention can generally be classified on the basis of thefollowing three groups:

-   i) The “GLEW-group”: NANOBODIES with the amino acid sequence GLEW    (SEQ ID NO: 817) at positions 44-47 according to the Kabat numbering    and Q at position 108 according to the Kabat numbering. As further    described herein, NANOBODIES within this group usually have a V at    position 37, and can have a W, P, R or S at position 103, and    preferably have a W at position 103. The GLEW: group also comprises    some GLEW-like sequences such as those mentioned in Table A-3 below.    More generally, and without limitation, NANOBODIES belonging to the    GLEW-group can be defined as NANOBODIES with a G at position 44    and/or with a W at position 47, in which position 46 is usually E    and in which preferably position 45 is not a charged amino acid    residue and not cysteine;-   ii) The “KERE-group”: NANOBODIES with the amino acid sequence KERE    (SEQ ID NO: 818) or KQRE (SEQ ID NO: 819) (or another KERE-like    sequence) at positions 43-46 according to the Kabat numbering and Q    or L at position 108 according to the Kabat numbering. As further    described herein, NANOBODIES within this group usually have a F at    position 37, an L or F at position 47; and can have a W, P, R or S    at position 103, and preferably have a W at position 103. More    generally, and without limitation, NANOBODIES belonging to the    KERE-group can be defined as NANOBODIES with a K, Q or R at position    44 (usually K) in which position 45 is a charged amino acid residue    or cysteine, and position 47 is as further defined herein;-   iii) The “103 P, R, S-group”: NANOBODIES with a P, R or S at    position 103. These NANOBODIES can have either the amino acid    sequence GLEW (SEQ ID NO: 817) at positions 44-47 according to the    Kabat numbering or the amino acid sequence KERE (SEQ ID NO: 818) or    KQRE (SEQ ID NO: 819) at positions 43-46 according to the Kabat    numbering, the latter most preferably in combination with an F at    position 37 and an L or an F at position 47 (as defined for the    KERE-group); and can have Q or L at position 108 according to the    Kabat numbering, and preferably have Q.

Also, where appropriate, NANOBODIES may belong to (i.e. havecharacteristics of) two or more of these classes. For example, onespecifically preferred group of NANOBODIES has GLEW (SEQ ID NO: 817) ora GLEW-like sequence at positions 44-47; P, R or S (and in particular R)at position 103; and Q at position 108 (which may be humanized to L).

More generally, it should be noted that the definitions referred toabove describe and apply to NANOBODIES in the form of a native (i.e.non-humanized) V_(HH) sequence, and that humanized variants of theseNANOBODIES may contain other amino acid residues than those indicatedabove (i.e. one or more humanizing substitutions as defined herein). Forexample, and without limitation, in some humanized NANOBODIES of theGLEW-group or the 103 P, R, S-group, Q at position 108 may be humanizedto 108L. As already mentioned herein, other humanizing substitutions(and suitable combinations thereof) will become clear to the skilledperson based on the disclosure herein. In addition, or alternatively,other potentially useful humanizing substitutions can be ascertained bycomparing the sequence of the framework regions of a naturally occurringV_(HH) sequence with the corresponding framework sequence of one or moreclosely related human V_(H) sequences, after which one or more of thepotentially useful humanizing substitutions (or combinations thereof)thus determined can be introduced into said V_(HH) sequence (in anymanner known per se, as further described herein) and the resultinghumanized V_(HH) sequences can be tested for affinity for the target,for stability, for ease and level of expression, and/or for otherdesired properties. In this way, by means of a limited degree of trialand error, other suitable humanizing substitutions (or suitablecombinations thereof) can be determined by the skilled person based onthe disclosure herein. Also, based on the foregoing, (the frameworkregions of) a NANOBODY may be partially humanized or fully humanized.

Thus, in another preferred, but non-limiting aspect, a NANOBODY of theinvention may be a NANOBODY belonging to the GLEW-group (as definedherein), and in which CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

In another preferred, but non-limiting aspect, a NANOBODY of theinvention may be a NANOBODY belonging to the KERE-group (as definedherein), and CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

Thus, in another preferred, but non-limiting aspect, a NANOBODY of theinvention may be a NANOBODY belonging to the 103 P, R, S-group (asdefined herein), and in which CDR1, CDR2 and CDR3 are as defined herein,and are preferably as defined according to one of the preferred aspectsherein, and are more preferably as defined according to one of the morepreferred aspects herein.

Also, more generally and in addition to the 108Q, 43E/44R and 103 P,R,Sresidues mentioned above, the NANOBODIES of the invention can contain,at one or more positions that in a conventional V_(H) domain would form(part of) the V_(H)/V_(L) interface, one or more amino acid residuesthat are more highly charged than the amino acid residues that naturallyoccur at the same position(s) in the corresponding naturally occurringV_(H) sequence, and in particular one or more charged amino acidresidues (as mentioned in Table A-2). Such substitutions include, butare not limited to, the GLEW-like sequences mentioned in Table A-3below; as well as the substitutions that are described in theInternational Application WO 00/29004 for so-called “microbodies”, e.g.so as to obtain a NANOBODY with Q at position 108 in combination withKLEW (SEQ ID NO: 850) at positions 44-47. Other possible substitutionsat these positions will be clear to the skilled person based upon thedisclosure herein.

In one aspect of the NANOBODIES of the invention, the amino acid residueat position 83 is chosen from the group consisting of L, M, S, V and W;and is preferably L.

Also, in one aspect of the NANOBODIES of the invention, the amino acidresidue at position 83 is chosen from the group consisting of R, K, N,E, G, I, T and Q; and is most preferably either K or E (for NANOBODIEScorresponding to naturally occurring V_(HH) domains) or R (for“humanized” NANOBODIES, as described herein). The amino acid residue atposition 84 is chosen from the group consisting of P, A, R, S, D T, andV in one aspect, and is most preferably P (for NANOBODIES correspondingto naturally occurring V_(HH) domains) or R (for “humanized” NANOBODIES,as described herein).

Furthermore, in one aspect of the NANOBODIES of the invention, the aminoacid residue at position 104 is chosen from the group consisting of Gand D; and is most preferably G.

Collectively, the amino acid residues at positions 11, 37, 44, 45, 47,83, 84, 103, 104 and 108, which in the NANOBODIES are as mentionedabove, will also be referred to herein as the “Hallmark Residues”. TheHallmark Residues and the amino acid residues at the correspondingpositions of the most closely related human V_(H) domain, V_(H)3, aresummarized in Table A-3.

Some especially preferred but non-limiting combinations of theseHallmark Residues as occur in naturally occurring V_(HH) domains arementioned in Table A-4. For comparison, the corresponding amino acidresidues of the human V_(H)3 called DP-47 have been indicated initalics.

TABLE A-3 Hallmark Residues in NANOBODIES Position Human V_(H)3 HallmarkResidues 11 L, V; L, M, S, V, W; preferably L predominantly L 37 V, I,F; usually V F⁽¹⁾, Y, H, I, L or V, preferably F⁽¹⁾ or Y    44⁽⁸⁾ GG⁽²⁾, E⁽³⁾, A, D, Q, R, S, L; preferably G⁽²⁾, E⁽³⁾ or Q; mostpreferably G⁽²⁾ or E⁽³⁾.    45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, C, I, L, P, Q, V;preferably L⁽²⁾ or R⁽³⁾    47⁽⁸⁾ W, Y W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R,S, V or Y; preferably W⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R 83 R or K; usually R R,K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, Q or T; preferably K or R; most preferably K 84A, T, D; P⁽⁵⁾, A, L, R, S, T, D, V; preferably P predominantly A 103  WW⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S; preferably W 104  G G or D; preferably G 108  L, Mor T; Q, L⁽⁷⁾ or R; preferably Q or L⁽⁷⁾ predominantly L Notes: ⁽¹⁾Inparticular, but not exclusively, in combination with KERE (SEQ ID NO:818) or KQRE (SEQ ID NO: 819) at positions 43-46. ⁽²⁾Usually as GLEW(SEQ ID NO: 817) at positions 44-47. ⁽³⁾Usually as KERE (SEQ ID NO: 818)or KQRE (SEQ ID NO: 819) at positions 43-46, e.g. as KEREL (SEQ ID NO:820), KEREF (SEQ ID NO: 821), KQREL (SEQ ID NO: 822), KQREF (SEQ ID NO:823) or KEREG (SEQ ID NO: 824) at positions 43-47. Alternatively, alsosequences such as TERE (SEQ ID NO: 825) (for example TEREL (SEQ ID NO:826)), KECE (SEQ ID NO: 827) (for example KECEL (SEQ ID NO: 828) orKECER (SEQ ID NO: 829)), RERE (SEQ ID NO: 830) (for example REREG (SEQID NO: 831)), QERE (SEQ ID NO: 832) (for example QEREG (SEQ ID NO:833)), KGRE (SEQ ID NO: 834) (for example KGREG (SEQ ID NO: 835)), KDRE(SEQ ID NO: 836) (for example KDREV (SEQ ID NO: 837)) are possible. Someother possible, but less preferred sequences include for example DECKL(SEQ ID NO: 838) and NVCEL (SEQ ID NO: 839). ⁽⁴⁾With both GLEW (SEQ IDNO: 817) at positions 44-47 and KERE (SEQ ID NO: 818) or KQRE (SEQ IDNO: 819) at positions 43-46. ⁽⁵⁾Often as KP or EP at positions 83-84 ofnaturally occurring V_(HH) domains. ⁽⁶⁾In particular, but notexclusively, in combination with GLEW (SEQ ID NO: 817) at positions44-47. ⁽⁷⁾With the proviso that when positions 44-47 are GLEW (SEQ IDNO: 817), position 108 is always Q in (non-humanized) V_(HH) sequencesthat also contain a W at 103. The GLEW-group also contains GLEW-likesequences at positions 44-47, such as for example GVEW (SEQ ID NO: 840),EPEW (SEQ ID NO: 841), GLER (SEQ ID NO: 842), DQEW (SEQ ID NO: 843),DLEW (SEQ ID NO: 844), GIEW (SEQ ID NO: 845), ELEW (SEQ ID NO: 846),GPEW (SEQ ID NO: 847), EWLP (SEQ ID NO: 848), and GPER (SEQ ID NO: 849.

TABLE A-4 Some preferred but non-limiting combinations of HallmarkResidues in naturally occurring NANOBODIES. For humanization of thesecombinations, reference is made to the specification. 11 37 44 45 47 8384 103 104 108 DP-47 (human) M V G L W R A W G L “KERE” group L F E R LK P W G Q L F E R F E P W G Q L F E R F K P W G Q L Y Q R L K P W G Q LF L R V K P Q G Q L F Q R L K P W G Q L F E R F K P W G Q “GLEW” group LV G L W K S W G Q M V G L W K P R G Q

In the NANOBODIES, each amino acid residue at any other position thanthe Hallmark Residues can be any amino acid residue that naturallyoccurs at the corresponding position (according to the Kabat numbering)of a naturally occurring V_(HH) domain.

Such amino acid residues will be clear to the skilled person. Tables A-5to A-8 mention some non-limiting residues that can be present at eachposition (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4of naturally occurring V_(HH) domains. For each position, the amino acidresidue that most frequently occurs at each position of a naturallyoccurring V_(HH) domain (and which is the most preferred amino acidresidue for said position in a NANOBODY) is indicated in bold; and otherpreferred amino acid residues for each position have been underlined(note: the number of amino acid residues that are found at positions26-30 of naturally occurring V_(HH) domains supports the hypothesisunderlying the numbering by Chothia (supra) that the residues at thesepositions already form part of CDR1).

In Tables A-5-A-8, some of the non-limiting residues that can be presentat each position of a human V_(H)3 domain have also been mentioned.Again, for each position, the amino acid residue that most frequentlyoccurs at each position of a naturally occurring human V_(H)3 domain isindicated in bold; and other preferred amino acid residues have beenunderlined.

For reference only, Tables A-5-A-8 also contain data on the V_(HH)entropy (“V_(HH) Ent.”) and V_(HH) variability (“V_(HH) Var.”) at eachamino acid position for a representative sample of 1118 V_(HH) sequences(data kindly provided by David Lutje Hulsing and Prof. Theo Verrips ofUtrecht University). The values for the V_(HH) entropy and the V_(HH)variability provide a measure for the variability and degree ofconservation of amino acid residues between the 1118 V_(HH) sequencesanalyzed: low values (i.e. <1, such as <0.5) indicate that an amino acidresidue is highly conserved between the V_(HH) sequences (i.e. littlevariability). For example, the G at position 8 and the G at position 9have values for the V_(HH) entropy of 0.1 and 0 respectively, indicatingthat these residues are highly conserved and have little variability(and in case of position 9 is G in all 1118 sequences analysed), whereasfor residues that form part of the CDR's generally values of 1.5 or moreare found (data not shown). Note that (1) the amino acid residues listedin the second column of Tables A-5-A-8 are based on a bigger sample thanthe 1118 V_(HH) sequences that were analysed for determining the V_(HH)entropy and V_(HH) variability referred to in the last two columns; and(2) the data represented below support the hypothesis that the aminoacid residues at positions 27-30 and maybe even also at positions 93 and94 already form part of the CDR's (although the invention is not limitedto any specific hypothesis or explanation, and as mentioned above,herein the numbering according to Kabat is used). For a generalexplanation of sequence entropy, sequence variability and themethodology for determining the same, see Oliveira et al., PROTEINS:Structure, Function and Genetics, 52: 544-552 (2003).

TABLE A-5 Non-limiting examples of amino acid residues in FR1 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'S Ent. Var. 1 E, Q Q, A, E — — 2V V 0.2 1 3 Q Q, K 0.3 2 4 L L 0.1 1 5 V, L Q, E, L, V 0.8 3 6 E E, D,Q, A 0.8 4 7 S, T S, F 0.3 2 8 G, R G 0.1 1 9 G G 0 1 10 G, V G, D, R0.3 2 11 Hallmark residue: L, M, S, V, W; preferably L 0.8 2 12 V, I V,A 0.2 2 13 Q, K, R Q, E, K, P, R 0.4 4 14 P A, Q, A, G, P, S, T, V 1 515 G G 0 1 16 G, R G, A, E, D 0.4 3 17 S S, F 0.5 2 18 L L, V 0.1 1 19R, K R, K, L, N, S, T 0.6 4 20 L L, F, I, V 0.5 4 21 S S, A, F, T 0.2 322 C C 0 1 23 A, T A, D, E, P, S, T, V 1.3 5 24 A A, I, L, S, T, V 1 625 S S, A, F, P, T 0.5 5 26 G G, A, D, E, R, S, T, V 0.7 7 27 F S, F, R,L, P, G, N, 2.3 13 28 T N, T, E, D, S, I, R, A, G, R, F, Y 1.7 11 29 F,V F, L, D, S, I, G, V, A 1.9 11 30 S, D, G N, S, E, G, A, D, M, T 1.8 11

TABLE A-6 Non-limiting examples of amino acid residues in FR2 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 36 W W 0.1 1 37Hallmark residue: F⁽¹⁾, H, I, 1.1 6 L, Y or V, preferably F⁽¹⁾ or Y 38 RR 0.2 1 39 Q Q, H, P, R 0.3 2 40 A A, F, G, L, P, T, V 0.9 7 41 P, S, TP, A, L, S 0.4 3 42 G G, E 0.2 2 43 K K, D, E, N, Q, R, T, V 0.7 6 44Hallmark residue: G⁽²⁾, E⁽³⁾, A, D, 1.3 5 Q, R, S, L; preferably G⁽²⁾,E⁽³⁾ or Q; most preferably G⁽²⁾ or E⁽³⁾. 45 Hallmark residue: L⁽²⁾,R⁽³⁾, C, 0.6 4 I, L, P, Q, V; preferably L⁽²⁾ or R⁽³⁾ 46 E, V E, D, K,Q, V 0.4 2 47 Hallmark residue: W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, 1.9 9 A, G, I, M, R,S, V or Y; preferably W⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R 48 V V, I, L 0.4 3 49 S, A,G A, S, G, T, V 0.8 3

TABLE A-7 Non-limiting examples of amino acid residues in FR3 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 66 R R 0.1 1 67 F F,L, V 0.1 1 68 T T, A, N, S 0.5 4 69 I I, L, M, V 0.4 4 70 S S, A, F, T0.3 4 71 R R, G, H, I, L, K, Q, S, T, W 1.2 8 72 D, E D, E, G, N, V 0.54 73 N, D, G N, A, D, F, I, K, L, R, S, T, V, Y 1.2 9 74 A, S A, D, G,N, P, S, T, V 1 7 75 K K, A, E, K, L, N, Q, R 0.9 6 76 N, S N, D, K, R,S, T, Y 0.9 6 77 S, T, I T, A, E, I, M, P, S 0.8 5 78 L, A V, L, A, F,G, I, M 1.2 5 79 Y, H Y, A, D, F, H, N, S, T 1 7 80 L L, F, V 0.1 1 81 QQ, E, I, L, R, T 0.6 5 82 M M, I, L, V 0.2 2 82a N, G N, D, G, H, S, T0.8 4 82b S S, N, D, G, R, T 1 6 82c L L, P, V 0.1 2 83 Hallmarkresidue: R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, Q 0.9 7 or T; preferably K or R;most preferably K 84 Hallmark residue: P⁽⁵⁾, A, D, L, R, 0.7 6 S, T, V;preferably P 85 E, G E, D, G, Q 0.5 3 86 D D 0 1 87 T, M T, A, S 0.2 388 A A, G, S 0.3 2 89 V, L V, A, D, I, L, M, N, R, T 1.4 6 90 Y Y, F 0 191 Y, H Y, D, F, H, L, S, T, V 0.6 4 92 C C 0 1 93 A, K, T A, N, G, H,K, N, R, S, T, V, Y 1.4 10 94 K, R, T A, V, C, F, G, I, K, L, R, S or T1.6 9

TABLE A-8 Non-limiting examples of amino acid residues in FR4 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 103 Hallmarkresidue: W⁽⁴⁾, P⁽⁶⁾, 0.4 2 R⁽⁶⁾, S; preferably W 104 Hallmark residue: Gor D; 0.1 1 preferably G 105 Q, R Q, E, K, P, R 0.6 4 106 G G 0.1 1 107T T, A, I 0.3 2 108 Hallmark residue: Q, L⁽⁷⁾ or 0.4 3 R; preferably Qor L⁽⁷⁾ 109 V V 0.1 1 110 T T, I, A 0.2 1 111 V V, A, I 0.3 2 112 S S, F0.3 1 113 S S, A, L, P, T 0.4 3

Thus, in another preferred, but not limiting aspect, a NANOBODY of theinvention can be defined as an amino acid sequence with the (general)structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) one or more of the amino acid residues at positions 11, 37, 44,    45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering    are chosen from the Hallmark residues mentioned in Table A-3;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above NANOBODIES may for example be V_(HH) sequences or may behumanized NANOBODIES. When the above NANOBODY sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the NANOBODIES are partially humanized NANOBODIES, they mayoptionally be further suitably humanized, again as described herein.

In particular, a NANOBODY of the invention can be an amino acid sequencewith the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) (preferably) one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 (it being understood that V_(HH) sequences will contain one or    more Hallmark residues; and that partially humanized NANOBODIES will    usually, and preferably, [still] contain one or more Hallmark    residues [although it is also within the scope of the invention to    provide—where suitable in accordance with the invention—partially    humanized NANOBODIES in which all Hallmark residues, but not one or    more of the other amino acid residues, have been humanized]; and    that in fully humanized NANOBODIES, where suitable in accordance    with the invention, all amino acid residues at the positions of the    Hallmark residues will be amino acid residues that occur in a human    V_(H)3 sequence. As will be clear to the skilled person based on the    disclosure herein that such V_(HH) sequences, such partially    humanized NANOBODIES with at least one Hallmark residue, such    partially humanized NANOBODIES without Hallmark residues and such    fully humanized NANOBODIES all form aspects of this invention);    and in which:-   ii) said amino acid sequence has at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 1 to    22, in which for the purposes of determining the degree of amino    acid identity, the amino acid residues that form the CDR sequences    (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are    disregarded;    and in which:-   iii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably    as defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above NANOBODIES may for example be V_(HH) sequences or may behumanized NANOBODIES. When the above NANOBODY sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the NANOBODIES are partially humanized NANOBODIES, they mayoptionally be further suitably humanized, again as described herein.

TABLE A-9 Representative amino acid sequences for NANOBODIES of theKERE, GLEW and P, R, S 103 group. The CDR's are indicated with XXXX KEREsequence no. 1 SEQ ID NO: 1EVQLVESGGGLVQPGGSLRLSCAASGIPFSXXXXXWFRQAPGKQRDSVAXXXXXRFTISRDNAKNTVYLQMNSLKPEDTAVYRCYFXXXXXWGQGTQVTVSS KERE sequence no. 2 SEQ IDNO: 2 QVKLEESGGGLVQAGGSLRLSCVGSGRTFSXXXXXWFRLAPGKEREFVAXXXXXRFTISRDTASNRGYLHMNNLTPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 3 SEQ IDNO: 3 AVQLVDSGGGLVQAGDSLKLSCALTGGAFTXXXXXWFRQTPGREREFVAXXXXXRFTISRDNAKNMVYLRMNSLIPEDAAVYSCAAXXXXXWGQGTLVTVSS KERE sequence no. 4 SEQ IDNO: 4 QVQLVESGGGLVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTISRDDAKNTVWLHGSTLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 5 SEQ IDNO: 5 AVQLVESGGGLVQGGGSLRLACAASERIFDXXXXXWYRQGPGNERELVAXXXXXRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKIXXXXXWGQGTQVTVSS KERE sequence no. 6 SEQ IDNO: 6 DVKFVESGGGLVQAGGSLRLSCVASGFNFDXXXXXWFRQAPGKEREEVAXXXXXRFTISSEKDKNSVYLQMNSLKPEDTALYICAGXXXXXWGRGTQVTVSS KERE sequence no. 7 SEQ IDNO: 7 QVRLAESGGGLVQSGGSLRLSCVASGSTYTXXXXXWYRQYPGKQRALVAXXXXXRFTIARDSTKDTFCLQMNNLKPEDTAVYYCYAXXXXXWGQGTQVTVSS KERE sequence no. 8 SEQ IDNO: 8 EVQLVESGGGLVQAGGSLRLSCAASGFTSDXXXXXWFRQAPGKPREGVSXXXXXRFTISTDNAKNTVHLLMNRVNAEDTALYYCAVXXXXXWGRGTRVTVSS KERE sequence no. 9 SEQ IDNO: 9 QVQLVESGGGLVQPGGSLRLSCQASGDISTXXXXXWYRQVPGKLREFVAXXXXXRFTISGDNAKRAIYLQMNNLKPDDTAVYYCNRXXXXXWGQGTQVTVSP KERE sequence no. 10 SEQ IDNO: 10 QVPVVESGGGLVQAGDSLRLFCAVPSFTSTXXXXXWFRQAPGKEREFVAXXXXXRFTISRNATKNTLTLRMDSLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 11 SEQ IDNO: 11 EVQLVESGGGLVQAGDSLRLFCTVSGGTASXXXXXWFRQAPGEKREFVAXXXXXRFTIARENAGNMVYLQMNNLKPDDTALYTCAAXXXXXWGRGTQVTVSS KERE sequence no. 12 SEQ IDNO: 12 AVQLVESGGDSVQPGDSQTLSCAASGRTNSXXXXXWFRQAPGKERVFLAXXXXXRFTISRDSAKNMMYLQMNNLKPQDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 13 SEQID NO: 13 AVQLVESGGGLVQAGGSLRLSCVVSGLTSSXXXXXWFRQTPWQERDFVAXXXXXRFTISRDNYKDTVLLEMNFLKPEDTAIYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 14 SEQID NO: 14 AVQLVESGGGLVQAGASLRLSCATSTRTLDXXXXXWFRQAPGRDREFVAXXXXXRFTVSRDSAENTVALQMNSLKPEDTAVYYCAAXXXXXWGQGTRVTVSS KERE sequence no. 15 SEQID NO: 15 QVQLVESGGGLVQPGGSLRLSCTVSRLTAHXXXXXWFRQAPGKEREAVSXXXXXRFTISRDYAGNTAFLQMDSLKPEDTGVYYCATXXXXXWGQGTQVTVSS KERE sequence no. 16 SEQ IDNO: 16 EVQLVESGGELVQAGGSLKLSCTASGRNFVXXXXXWFRRAPGKEREFVAXXXXXRFTVSRDNGKNTAYLRMNSLKPEDTADYYCAVXXXXXLGSGTQVTVSS GLEW sequence no. 1 SEQ IDNO: 17 AVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKVLEWVSXXXXXRFTISRDNAKNTLYLQMNSLKPEDTAVYYCVKXXXXXGSQGTQVTVSS GLEW sequence no. 2 SEQ IDNO: 18 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS GLEW sequence no. 3 SEQID NO: 19 EVQLVESGGGLALPGGSLTLSCVFSGSTFSXXXXXWVRHTPGKAEEWVSXXXXXRFTISRDNAKNTLYLEMNSLSPEDTAMYYCGRXXXXXRSKGIQVTVSS P, R, S 103 sequence SEQ IDNO: 20 AVQLVESGGGLVQAGGSLRLSCAASGRTFSXXXXXWFRQAPGKEREFVAXXXXXRFTI no. 1SRDNAKNTVYLQMNSLKPEDTAVYYCAAXXXXXRGQGTQVTVSS P, R, S 103 sequence SEQ IDNO: 21 DVQLVESGGDLVQPGGSLRLSCAASGFSFDXXXXXWLRQTPGKGLEWVGXXXXXRFT no. 2ISRDNAKNMLYLHLNNLKSEDTAVYYCRRXXXXXLGQGTQVTVSS P, R, S 103 sequence SEQID NO: 22 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRF no. 3KISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS

In particular, a NANOBODY of the invention of the KERE group can be anamino acid sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which:

-   i) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid (as defined herein) or a cysteine    residue, and position 44 is preferably an E;    and in which:

-   ii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-10 Representative FW1 sequences for NANOBODIES of theKERE-group. KERE FW1 sequence no. 1 SEQ ID NO: 23QVQRVESGGGLVQAGGSLRLSCAASGRTSS KERE FW1 sequence no. 2 SEQ ID NO: 24QVQLVESGGGLVQTGDSLSLSCSASGRTFS KERE FW1 sequence no. 3 SEQ ID NO: 25QVKLEESGGGLVQAGDSLRLSCAATGRAFG KERE FW1 sequence no. 4 SEQ ID NO: 26AVQLVESGGGLVQPGESLGLSCVASGRDFV KERE FW1 sequence no. 5 SEQ ID NO: 27EVQLVESGGGLVQAGGSLRLSCEVLGRTAG KERE FW1 sequence no. 6 SEQ ID NO: 28QVQLVESGGGWVQPGGSLRLSCAASETILS KERE FW1 sequence no. 7 SEQ ID NO: 29QVQLVESGGGTVQPGGSLNLSCVASGNTFN KERE FW1 sequence no. 8 SEQ ID NO: 30EVQLVESGGGLAQPGGSLQLSCSAPGFTLD KERE FW1 sequence no. 9 SEQ ID NO: 31AQELEESGGGLVQAGGSLRLSCAASGRTFNand in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-11 Representative FW2 sequences for NANOBODIES of theKERE-group. KERE FW2 sequence SEQ ID NO: 41 WFRQAPGKEREFVA no. 1 KEREFW2 sequence SEQ ID NO: 42 WFRQTPGREREFVA no. 2 KERE FW2 sequence SEQ IDNO: 43 WYRQAPGKQREMVA no. 3 KERE FW2 sequence SEQ ID NO: 44WYRQGPGKQRELVA no. 4 KERE FW2 sequence SEQ ID NO: 45 WIRQAPGKEREGVS no.5 KERE FW2 sequence SEQ ID NO: 46 WFREAPGKEREGIS no. 6 KERE FW2 sequenceSEQ ID NO: 47 WYRQAPGKERDLVA no. 7 KERE FW2 sequence SEQ ID NO: 48WFRQAPGKQREEVS no. 8 KERE FW2 sequence SEQ ID NO: 49 WFRQPPGKVREFVG no.9and in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-12 Representative FW3 sequences for NANOBODIES of theKERE-group. KERE FW3 sequence no. 1 SEQ ID NO: 50RFTISRDNAKNTVYLQMNSLKPEDTAVYRCYF KERE FW3 sequence no. 2 SEQ ID NO: 51RFAISRDNNKNTGYLQMNSLEPEDTAVYYCAA KERE FW3 sequence no. 3 SEQ ID NO: 52RFTVARNNAKNTVNLEMNSLKPEDTAVYYCAA KERE FW3 sequence no. 4 SEQ ID NO: 53RFTISRDIAKNTVDLLMNNLEPEDTAVYYCAA KERE FW3 sequence no. 5 SEQ ID NO: 54RLTISRDNAVDTMYLQMNSLKPEDTAVYYCAA KERE FW3 sequence no. 6 SEQ ID NO: 55RFTISRDNAKNTVYLQMDNVKPEDTAIYYCAA KERE FW3 sequence no. 7 SEQ ID NO: 56RFTISKDSGKNTVYLQMTSLKPEDTAVYYCAT KERE FW3 sequence no. 8 SEQ ID NO: 57RFTISRDSAKNMMYLQMNNLKPQDTAVYYCAA KERE FW3 sequence no. 9 SEQ ID NO: 58RFTISRENDKSTVYLQLNSLKPEDTAVYYCAA KERE FW3 sequence no. 10 SEQ ID NO: 59RFTISRDYAGNTAYLQMNSLKPEDTGVYYCATand in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-13 Representative FW4 sequences for NANOBODIES of theKERE-group. KERE FW4 sequence no. 1 SEQ ID NO: 60 WGQGTQVTVSS KERE FW4sequence no. 2 SEQ ID NO: 61 WGKGTLVTVSS KERE FW4 sequence no. 3 SEQ IDNO: 62 RGQGTRVTVSS KERE FW4 sequence no. 4 SEQ ID NO: 63 WGLGTQVTISSand in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above NANOBODIES, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized NANOBODIES).

Also, the above NANOBODIES may for example be V_(HH) sequences or may behumanized NANOBODIES. When the above NANOBODY sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the NANOBODIES are partially humanized NANOBODIES, they mayoptionally be further suitably humanized, again as described herein.

With regard to framework 1, it will be clear to the skilled person that,when an amino acid sequence as outlined above is generated by expressionof a nucleotide sequence, the first four amino acid sequences (i e aminoacid residues 1-4 according to the Kabat numbering) may often bedetermined by the primer(s) that have been used to generate said nucleicacid. Thus, for determining the degree of amino acid identity, the firstfour amino acid residues are preferably disregarded.

Also, with regard to framework 1, and although amino acid positions 27to 30 are according to the Kabat numbering considered to be part of theframework regions (and not the CDR's), it has been found by analysis ofa database of more than 1000 V_(HH) sequences that the positions 27 to30 have a variability (expressed in terms of V_(HH) entropy and V_(HH)variability—see Tables A-5 to A-8) that is much greater than thevariability on positions 1 to 26. Because of this, for determining thedegree of amino acid identity, the amino acid residues at positions 27to 30 are preferably also disregarded.

In view of this, a NANOBODY of the KERE class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid (as defined herein) or a cysteine    residue, and position 44 is preferably an E;    and in which:-   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE A-14 Representative FW1 sequences (amino acid residues 5 to 26)for NANOBODIES of the KERE-group. KERE FW1 sequence no. 10 SEQ ID NO: 32VESGGGLVQPGGSLRLSCAASG KERE FW1 sequence no. 11 SEQ ID NO: 33VDSGGGLVQAGDSLKLSCALTG KERE FW1 sequence no. 12 SEQ ID NO: 34VDSGGGLVQAGDSLRLSCAASG KERE FW1 sequence no. 13 SEQ ID NO: 35VDSGGGLVEAGGSLRLSCQVSE KERE FW1 sequence no. 14 SEQ ID NO: 36QDSGGGSVQAGGSLKLSCAASG KERE FW1 sequence no. 15 SEQ ID NO: 37VQSGGRLVQAGDSLRLSCAASE KERE FW1 sequence no. 16 SEQ ID NO: 38VESGGTLVQSGDSLKLSCASST KERE FW1 sequence no. 17 SEQ ID NO: 39MESGGDSVQSGGSLTLSCVASG KERE FW1 sequence no. 18 SEQ ID NO: 40QASGGGLVQAGGSLRLSCSASVand in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4    of NANOBODIES of the KERE-class;    and in which:-   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above NANOBODIES may for example be V_(HH) sequences or may behumanized NANOBODIES. When the above NANOBODY sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the NANOBODIES are partially humanized NANOBODIES, they mayoptionally be further suitably humanized, again as described herein.

A NANOBODY of the GLEW class may be an amino acid sequence that iscomprised of four framework regions/sequences interrupted by threecomplementarity determining regions/sequences, in which

-   i) preferably, when the NANOBODY of the GLEW-class is a    non-humanized NANOBODY, the amino acid residue in position 108 is Q;-   ii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-15 Representative FW1 sequences for NANOBODIES of theGLEW-group. GLEW FW1 sequence no. 1 SEQ ID NO: 64QVQLVESGGGLVQPGGSLRLSCAASGFTFS GLEW FW1 sequence no. 2 SEQ ID NO: 65EVHLVESGGGLVRPGGSLRLSCAAFGFIFK GLEW FW1 sequence no. 3 SEQ ID NO: 66QVKLEESGGGLAQPGGSLRLSCVASGFTFS GLEW FW1 sequence no. 4 SEQ ID NO: 67EVQLVESGGGLVQPGGSLRLSCVCVSSGCT GLEW FW1 sequence no. 5 SEQ ID NO: 68EVQLVESGGGLALPGGSLTLSCVFSGSTFSand in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-16 Representative FW2 sequences for NANOBODIES of theGLEW-group. GLEW FW2 sequence no. SEQ ID NO: 72 WVRQAPGKVLEWVS 1 GLEWFW2 sequence no. SEQ ID NO: 73 WVRRPPGKGLEWVS 2 GLEW FW2 sequence no.SEQ ID NO: 74 WVRQAPGMGLEWVS 3 GLEW FW2 sequence no. SEQ ID NO: 75WVRQAPGKEPEWVS 4 GLEW FW2 sequence no. SEQ ID NO: 76 WVRQAPGKDQEWVS 5GLEW FW2 sequence no. SEQ ID NO: 77 WVRQAPGKAEEWVS 6 GLEW FW2 sequenceno. SEQ ID NO: 78 WVRQAPGKGLEWVA 7 GLEW FW2 sequence no. SEQ ID NO: 79WVRQAPGRATEWVS 8and in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-17 Representative FW3 sequences for NANOBODIES of theGLEW-group. GLEW FW3 sequence no. 1 SEQ ID NO: 80RFTISRDNAKNTLYLQMNSLKPEDTAVYYCVK GLEW FW3 sequence no. 2 SEQ ID NO: 81RFTISRDNARNTLYLQMDSLIPEDTALYYCAR GLEW FW3 sequence no. 3 SEQ ID NO: 82RFTSSRDNAKSTLYLQMNDLKPEDTALYYCAR GLEW FW3 sequence no. 4 SEQ ID NO: 83RFIISRDNAKNTLYLQMNSLGPEDTAMYYCQR GLEW FW3 sequence no. 5 SEQ ID NO: 84RFTASRDNAKNTLYLQMNSLKSEDTARYYCAR GLEW FW3 sequence no. 6 SEQ ID NO: 85RFTISRDNAKNTLYLQMDDLQSEDTAMYYCGRand in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-18 Representative FW4 sequences for NANOBODIES of theGLEW-group. GLEW FW4 sequence no. 1 SEQ ID NO: 86 GSQGTQVTVSS GLEW FW4sequence no. 2 SEQ ID NO: 87 LRGGTQVTVSS GLEW FW4 sequence no. 3 SEQ IDNO: 88 RGQGTLVTVSS GLEW FW4 sequence no. 4 SEQ ID NO: 89 RSRGIQVTVSSGLEW FW4 sequence no. 5 SEQ ID NO: 90 WGKGTQVTVSS GLEW FW4 sequence no.6 SEQ ID NO: 91 WGQGTQVTVSSand in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above NANOBODIES, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized NANOBODIES).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a NANOBODY of the GLEW class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) preferably, when the NANOBODY of the GLEW-class is a    non-humanized NANOBODY, the amino acid residue in position 108 is Q;    and in which:-   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE A-19 Representative FW1 sequences (amino acid residues 5 to 26)for NANOBODIES of the KERE-group. GLEW FW1 sequence no. 6 SEQ ID NO: 69VESGGGLVQPGGSLRLSCAASG GLEW FW1 sequence no. 7 SEQ ID NO: 70EESGGGLAQPGGSLRLSCVASG GLEW FW1 sequence no. 8 SEQ ID NO: 71VESGGGLALPGGSLTLSCVFSGand in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4    of NANOBODIES of the GLEW-class;    and in which:-   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above NANOBODIES may for example be V_(HH) sequences or may behumanized NANOBODIES. When the above NANOBODY sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the NANOBODIES are partially humanized NANOBODIES, they mayoptionally be further suitably humanized, again as described herein. Inthe above NANOBODIES, one or more of the further Hallmark residues arepreferably as described herein (for example, when they are V_(HH)sequences or partially humanized NANOBODIES).

A NANOBODY of the P, R, S 103 class may be an amino acid sequence thatis comprised of four framework regions/sequences interrupted by threecomplementarity determining regions/sequences, in which

-   i) the amino acid residue at position 103 according to the Kabat    numbering is different from W;    and in which:-   ii) preferably the amino acid residue at position 103 according to    the Kabat numbering is P, R or S, and more preferably R;    and in which:-   iii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-20 Representative FW1 sequences for NANOBODIES of the P, R, S103-group. P, R, S 103 FW1 sequence no. 1 SEQ ID NO: 92AVQLVESGGGLVQAGGSLRLSCAASGRTFS P, R, S 103 FW1 sequence no. 2 SEQ ID NO:93 QVQLQESGGGMVQPGGSLRLSCAASGFDFG P, R, S 103 FW1 sequence no. 3 SEQ IDNO: 94 EVHLVESGGGLVRPGGSLRLSCAAFGFIFK P, R, S 103 FW1 sequence no. 4 SEQID NO: 95 QVQLAESGGGLVQPGGSLKLSCAASRTIVS P, R, S 103 FW1 sequence no. 5SEQ ID NO: 96 QEHLVESGGGLVDIGGSLRLSCAASERIFS P, R, S 103 FW1 sequenceno. 6 SEQ ID NO: 97 QVKLEESGGGLAQPGGSLRLSCVASGFTFS P, R, S 103 FW1sequence no. 7 SEQ ID NO: 98 EVQLVESGGGLVQPGGSLRLSCVCVSSGCT P, R, S 103FW1 sequence no. 8 SEQ ID NO: 99 EVQLVESGGGLALPGGSLTLSCVFSGSTFSand in which

-   iv) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-21 Representative FW2 sequences for NANOBODIES of the P, R, S103-group. P, R, S 103 FW2 sequence no. 1 SEQ ID NO: 102 WFRQAPGKEREFVAP, R, S 103 FW2 sequence no. 2 SEQ ID NO: 103 WVRQAPGKVLEWVS P, R, S 103FW2 sequence no. 3 SEQ ID NO: 104 WVRRPPGKGLEWVS P, R, S 103 FW2sequence no. 4 SEQ ID NO: 105 WIRQAPGKEREGVS P, R, S 103 FW2 sequenceno. 5 SEQ ID NO: 106 WVRQYPGKEPEWVS P, R, S 103 FW2 sequence no. 6 SEQID NO: 107 WFRQPPGKEHEFVA P, R, S 103 FW2 sequence no. 7 SEQ ID NO: 108WYRQAPGKRTELVA P, R, S 103 FW2 sequence no. 8 SEQ ID NO: 109WLRQAPGQGLEWVS P, R, S 103 FW2 sequence no. 9 SEQ ID NO: 110WLRQTPGKGLEWVG P, R, S 103 FW2 sequence no. 10 SEQ ID NO: 111WVRQAPGKAEEFVSand in which:

-   v) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-22 Representative FW3 sequences for NANOBODIES of the P, R, S103-group. P, R, S 103 FW3 sequence no. 1 SEQ ID NO: 112RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA P, R, S 103 FW3 sequence no. 2 SEQ IDNO: 113 RFTISRDNARNTLYLQMDSLIPEDTALYYCAR P, R, S 103 FW3 sequence no. 3SEQ ID NO: 114 RFTISRDNAKNEMYLQMNNLKTEDTGVYWCGA P, R, S 103 FW3 sequenceno. 4 SEQ ID NO: 115 RFTISSDSNRNMIYLQMNNLKPEDTAVYYCAA P, R, S 103 FW3sequence no. 5 SEQ ID NO: 116 RFTISRDNAKNMLYLHLNNLKSEDTAVYYCRR P, R, S103 FW3 sequence no. 6 SEQ ID NO: 117 RFTISRDNAKKTVYLRLNSLNPEDTAVYSCNLP, R, S 103 FW3 sequence no. 7 SEQ ID NO: 118RFKISRDNAKKTLYLQMNSLGPEDTAMYYCQR P, R, S 103 FW3 sequence no. 8 SEQ IDNO: 119 RFTVSRDNGKNTAYLRMNSLKPEDTADYYCAVand in which:

-   vi) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-23 Representative FW4 sequences for NANOBODIES of the P, R, S103-group. P, R, S 103 FW4 sequence SEQ ID NO: 120 RGQGTQVTVSS no. 1 P,R, S 103 FW4 sequence SEQ ID NO: 121 LRGGTQVTVSS no. 2 P, R, S 103 FW4sequence SEQ ID NO: 122 GNKGTLVTVSS no. 3 P, R, S 103 FW4 sequence SEQID NO: 123 SSPGTQVTVSS no. 4 P, R, S 103 FW4 sequence SEQ ID NO: 124SSQGTLVTVSS no. 5 P, R, S 103 FW4 sequence SEQ ID NO: 125 RSRGIQVTVSSno. 6and in which:

-   vii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably    as defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above NANOBODIES, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized NANOBODIES).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a NANOBODY of the P,R,S 103 class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) the amino acid residue at position 103 according to the Kabat    numbering is different from W;    and in which:-   ii) preferably the amino acid residue at position 103 according to    the Kabat numbering is P, R or S, and more preferably R;    and in which:    iii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE A-24 Representative FW1 sequences (amino acid residues 5 to 26)for NANOBODIES of the P, R, S 103-group. P, R, S 103 FW1 SEQ ID NO:VESGGGLVQAGGSLRLSCAASG sequence no. 9 100 P, R, S 103 FW1 SEQ ID NO:AESGGGLVQPGGSLKLSCAASR sequence no. 10 101and in which:

-   iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of    NANOBODIES of the P,R,S 103 class;    and in which:-   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above NANOBODIES may for example be V_(HH) sequences or may behumanized NANOBODIES. When the above NANOBODY sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the NANOBODIES are partially humanized NANOBODIES, they mayoptionally be further suitably humanized, again as described herein.

In the above NANOBODIES, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized NANOBODIES).

In another preferred, but non-limiting aspect, the invention relates toa NANOBODY as described above, in which the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 560-621. This degree of amino acid identity can for example bedetermined by determining the degree of amino acid identity (in a mannerdescribed herein) between said NANOBODY and one or more of the sequencesof SEQ ID NO's: 560-621, in which the amino acid residues that form theframework regions are disregarded. Such NANOBODIES can be as furtherdescribed herein.

As already mentioned herein, another preferred but non-limiting aspectof the invention relates to a NANOBODY with an amino acid sequence thatis chosen from the group consisting of SEQ ID NO's: 560-621 or from thegroup consisting of from amino acid sequences that have more than 80%,preferably more than 90%, more preferably more than 95%, such as 99% ormore sequence identity (as defined herein) with at least one of theamino acid sequences of SEQ ID NO's: 560-621.

Also, in the above NANOBODIES:

-   i) any amino acid substitution (when it is not a humanizing    substitution as defined herein) is preferably, and compared to the    corresponding amino acid sequence of SEQ ID NO's: 560-621, a    conservative amino acid substitution, (as defined herein);    and/or:-   ii) its amino acid sequence preferably contains either only amino    acid substitutions, or otherwise preferably no more than 5,    preferably no more than 3, and more preferably only 1 or 2 amino    acid deletions or insertions, compared to the corresponding amino    acid sequence of SEQ ID NO's: 560-621;    and/or-   iii) the CDR's may be CDR's that are derived by means of affinity    maturation, for example starting from the CDR's of to the    corresponding amino acid sequence of SEQ ID NO's: 560-621.

Preferably, the CDR sequences and FR sequences in the NANOBODIES of theinvention are such that the NANOBODIES of the invention (andpolypeptides of the invention comprising the same):

-   -   bind to RANK-L with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to RANK-L with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more        preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between        10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to RANK-L with a k_(off) rate between 1 s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶        s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, CDR sequences and FR sequences present in the NANOBODIES ofthe invention are such that the NANOBODIES of the invention will bind toRANK-L with an affinity less than 500 nM, preferably less than 200 nM,more preferably less than 10 nM, such as less than 500 pM.

According to one non-limiting aspect of the invention, a NANOBODY may beas defined herein, but with the proviso that it has at least “one aminoacid difference” (as defined herein) in at least one of the frameworkregions compared to the corresponding framework region of a naturallyoccurring human V_(H) domain, and in particular compared to thecorresponding framework region of DP-47. More specifically, according toone non-limiting aspect of the invention, a NANOBODY may be as definedherein, but with the proviso that it has at least “one amino aciddifference” (as defined herein) at at least one of the Hallmark residues(including those at positions 108, 103 and/or 45) compared to thecorresponding framework region of a naturally occurring human V_(H)domain, and in particular compared to the corresponding framework regionof DP-47. Usually, a NANOBODY will have at least one such amino aciddifference with a naturally occurring V_(H) domain in at least one ofFR2 and/or FR4, and in particular at at least one of the Hallmarkresidues in FR2 and/or FR4 (again, including those at positions 108, 103and/or 45).

Also, a humanized NANOBODY of the invention may be as defined herein,but with the proviso that it has at least “one amino acid difference”(as defined herein) in at least one of the framework regions compared tothe corresponding framework region of a naturally occurring V_(HH)domain. More specifically, according to one non-limiting aspect of theinvention, a humanized NANOBODY may be as defined herein, but with theproviso that it has at least “one amino acid difference” (as definedherein) at at least one of the Hallmark residues (including those atpositions 108, 103 and/or 45) compared to the corresponding frameworkregion of a naturally occurring V_(HH) domain Usually, a humanizedNANOBODY will have at least one such amino acid difference with anaturally occurring V_(HH) domain in at least one of FR2 and/or FR4, andin particular at at least one of the Hallmark residues in FR2 and/or FR4(again, including those at positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is also within the scopeof the invention to use natural or synthetic analogs, mutants, variants,alleles, homologs and orthologs (herein collectively referred to as“analogs”) of the NANOBODIES of the invention as defined herein, and inparticular analogs of the NANOBODIES of SEQ ID NO's: 560-621. Thus,according to one aspect of the invention, the term “NANOBODY of theinvention” in its broadest sense also covers such analogs.

Generally, in such analogs, one or more amino acid residues may havebeen replaced, deleted and/or added, compared to the NANOBODIES of theinvention as defined herein. Such substitutions, insertions or deletionsmay be made in one or more of the framework regions and/or in one ormore of the CDR's. When such substitutions, insertions or deletions aremade in one or more of the framework regions, they may be made at one ormore of the Hallmark residues and/or at one or more of the otherpositions in the framework residues, although substitutions, insertionsor deletions at the Hallmark residues are generally less preferred(unless these are suitable humanizing substitutions as describedherein).

By means of non-limiting examples, a substitution may for example be aconservative substitution (as described herein) and/or an amino acidresidue may be replaced by another amino acid residue that naturallyoccurs at the same position in another V_(HH) domain (see Tables A-5 toA-8 for some non-limiting examples of such substitutions), although theinvention is generally not limited thereto. Thus, any one or moresubstitutions, deletions or insertions, or any combination thereof, thateither improve the properties of the NANOBODY of the invention or thatat least do not detract too much from the desired properties or from thebalance or combination of desired properties of the NANOBODY of theinvention (i.e. to the extent that the NANOBODY is no longer suited forits intended use) are included within the scope of the invention. Askilled person will generally be able to determine and select suitablesubstitutions, deletions or insertions, or suitable combinations ofthereof, based on the disclosure herein and optionally after a limiteddegree of routine experimentation, which may for example involveintroducing a limited number of possible substitutions and determiningtheir influence on the properties of the NANOBODIES thus obtained.

For example, and depending on the host organism used to express theNANOBODY or polypeptide of the invention, such deletions and/orsubstitutions may be designed in such a way that one or more sites forpost-translational modification (such as one or more glycosylationsites) are removed, as will be within the ability of the person skilledin the art. Alternatively, substitutions or insertions may be designedso as to introduce one or more sites for attachment of functional groups(as described herein), for example to allow site-specific pegylation(again as described herein).

As can be seen from the data on the V_(HH) entropy and V_(HH)variability given in Tables A-5 to A-8 above, some amino acid residuesin the framework regions are more conserved than others. Generally,although the invention in its broadest sense is not limited thereto, anysubstitutions, deletions or insertions are preferably made at positionsthat are less conserved. Also, generally, amino acid substitutions arepreferred over amino acid deletions or insertions.

The analogs are preferably such that they can bind to RANK-L with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein for the NANOBODIES of the invention.

The analogs are preferably also such that they retain the favourableproperties the NANOBODIES, as described herein.

Also, according to one preferred aspect, the analogs have a degree ofsequence identity of at least 70%, preferably at least 80%, morepreferably at least 90%, such as at least 95% or 99% or more; and/orpreferably have at most 20, preferably at most 10, even more preferablyat most 5, such as 4, 3, 2 or only 1 amino acid difference (as definedherein), with one of the NANOBODIES of SEQ ID NOs: 560-621.

Also, the framework sequences and CDR's of the analogs are preferablysuch that they are in accordance with the preferred aspects definedherein. More generally, as described herein, the analogs will have (a) aQ at position 108; and/or (b) a charged amino acid or a cysteine residueat position 45 and preferably an E at position 44, and more preferably Eat position 44 and R at position 45; and/or (c) P, R or S at position103.

One preferred class of analogs of the NANOBODIES of the inventioncomprise NANOBODIES that have been humanized (i.e. compared to thesequence of a naturally occurring NANOBODY of the invention). Asmentioned in the background art cited herein, such humanizationgenerally involves replacing one or more amino acid residues in thesequence of a naturally occurring V_(HH) with the amino acid residuesthat occur at the same position in a human V_(H) domain, such as a humanV_(H)3 domain. Examples of possible humanizing substitutions orcombinations of humanizing substitutions will be clear to the skilledperson, for example from the Tables herein, from the possible humanizingsubstitutions mentioned in the background art cited herein, and/or froma comparison between the sequence of a NANOBODY and the sequence of anaturally occurring human V_(H) domain.

The humanizing substitutions should be chosen such that the resultinghumanized NANOBODIES still retain the favourable properties ofNANOBODIES as defined herein, and more preferably such that they are asdescribed for analogs in the preceding paragraphs. A skilled person willgenerally be able to determine and select suitable humanizingsubstitutions or suitable combinations of humanizing substitutions,based on the disclosure herein and optionally after a limited degree ofroutine experimentation, which may for example involve introducing alimited number of possible humanizing substitutions and determiningtheir influence on the properties of the NANOBODIES thus obtained.

Generally, as a result of humanization, the NANOBODIES of the inventionmay become more “human-like”, while still retaining the favorableproperties of the NANOBODIES of the invention as described herein. As aresult, such humanized NANOBODIES may have several advantages, such as areduced immunogenicity, compared to the corresponding naturallyoccurring V_(HH) domains. Again, based on the disclosure herein andoptionally after a limited degree of routine experimentation, theskilled person will be able to select humanizing substitutions orsuitable combinations of humanizing substitutions which optimize orachieve a desired or suitable balance between the favourable propertiesprovided by the humanizing substitutions on the one hand and thefavourable properties of naturally occurring V_(HH) domains on the otherhand.

The NANOBODIES of the invention may be suitably humanized at anyframework residue(s), such as at one or more Hallmark residues (asdefined herein) or at one or more other framework residues (i.e.non-Hallmark residues) or any suitable combination thereof. Onepreferred humanizing substitution for NANOBODIES of the “P,R,S-103group” or the “KERE group” is Q108 into L108. NANOBODIES of the “GLEWclass” may also be humanized by a Q108 into L108 substitution, providedat least one of the other Hallmark residues contains a camelid(camelizing) substitution (as defined herein). For example, as mentionedabove, one particularly preferred class of humanized NANOBODIES has GLEWor a GLEW-like sequence at positions 44-47; P, R or S (and in particularR) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding thesame, can be provided in any manner known per se. For example, theanalogs can be obtained by providing a nucleic acid that encodes anaturally occurring V_(HH) domain, changing the codons for the one ormore amino acid residues that are to be substituted into the codons forthe corresponding desired amino acid residues (e.g. by site-directedmutagenesis or by PCR using suitable mismatch primers), expressing thenucleic acid/nucleotide sequence thus obtained in a suitable host orexpression system; and optionally isolating and/or purifying the analogthus obtained to provide said analog in essentially isolated form (e.g.as further described herein). This can generally be performed usingmethods and techniques known per se, which will be clear to the skilledperson, for example from the handbooks and references cited herein, thebackground art cited herein and/or from the further description herein.Alternatively, a nucleic acid encoding the desired analog can besynthesized in a manner known per se (for example using an automatedapparatus for synthesizing nucleic acid sequences with a predefinedamino acid sequence) and can then be expressed as described herein. Yetanother technique may involve combining one or more naturally occurringand/or synthetic nucleic acid sequences each encoding a part of thedesired analog, and then expressing the combined nucleic acid sequenceas described herein. Also, the analogs can be provided using chemicalsynthesis of the pertinent amino acid sequence using techniques forpeptide synthesis known per se, such as those mentioned herein.

In this respect, it will be also be clear to the skilled person that theNANOBODIES of the invention (including their analogs) can be designedand/or prepared starting from human V_(H) sequences (i e amino acidsequences or the corresponding nucleotide sequences), such as forexample from human V_(H)3 sequences such as DP-47, DP-51 or DP-29, i.e.by introducing one or more camelizing substitutions (i.e. changing oneor more amino acid residues in the amino acid sequence of said humanV_(H) domain into the amino acid residues that occur at thecorresponding position in a V_(HH) domain), so as to provide thesequence of a NANOBODY of the invention and/or so as to confer thefavourable properties of a NANOBODY to the sequence thus obtained.Again, this can generally be performed using the various methods andtechniques referred to in the previous paragraph, using an amino acidsequence and/or nucleotide sequence for a human V_(H) domain as astarting point.

Some preferred, but non-limiting camelizing substitutions can be derivedfrom Tables A-5-A-8. It will also be clear that camelizing substitutionsat one or more of the Hallmark residues will generally have a greaterinfluence on the desired properties than substitutions at one or more ofthe other amino acid positions, although both and any suitablecombination thereof are included within the scope of the invention. Forexample, it is possible to introduce one or more camelizingsubstitutions that already confer at least some the desired properties,and then to introduce further camelizing substitutions that eitherfurther improve said properties and/or confer additional favourableproperties. Again, the skilled person will generally be able todetermine and select suitable camelizing substitutions or suitablecombinations of camelizing substitutions, based on the disclosure hereinand optionally after a limited degree of routine experimentation, whichmay for example involve introducing a limited number of possiblecamelizing substitutions and determining whether the favourableproperties of NANOBODIES are obtained or improved (i.e. compared to theoriginal V_(H) domain).

Generally, however, such camelizing substitutions are preferably suchthat the resulting an amino acid sequence at least contains (a) a Q atposition 108; and/or (b) a charged amino acid or a cysteine residue atposition 45 and preferably also an E at position 44, and more preferablyE at position 44 and R at position 45; and/or (c) P, R or S at position103; and optionally one or more further camelizing substitutions. Morepreferably, the camelizing substitutions are such that they result in aNANOBODY of the invention and/or in an analog thereof (as definedherein), such as in a humanized analog and/or preferably in an analogthat is as defined in the preceding paragraphs.

Other analogs and nucleic acid sequences encoding the same, can beprovided, for example to improve stability of the NANOBODY. Duringstorage, NANOBODIES and other types of immunoglobulins may generatecertain variants as a result of:

-   i) oxidation event(s), occurring in typically only the “accessible”    methionines wherein oxidation increases during storage in parallel    with incubation temperature and time;-   ii) cyclization of the first glutamic acid residue, if present,    resulting in formation of pyroglutamate, and-   iii) isomerization of only the “accessible” aspartic acids or    asparagines in a DG, DS, NG or NS motif wherein isomerization    increases during storage in parallel with incubation temperature and    time.    Analogs of variants of the NANOBODIES of the invention may be    generated that have improved stability profile. This can be done,    for example, but without being limiting, by avoiding isomerization    of Asp (D) and Asn (N), e.g. by replacing the Asp-Gly (DG), Asp-Ser    (DS), Asn-Gly (NG) and Asn-Ser (NS) in the CDRs with another amino    acid such as e.g. Glu (E) or Gln (Q); by avoiding oxidation of Met    e.g. by replacing Met which are susceptible to forced oxidation with    another amino acid such as e.g. an Ala or Thr, and/or by replacing    N-terminal Glu by an alternative N-terminus, e.g. Asp. Again, the    skilled person will generally be able to determine and select    suitable stabilizing substitutions or suitable combinations of    stabilizing substitutions, based on the disclosure herein and    optionally after a limited degree of routine experimentation, which    may for example involve introducing a limited number of possible    stabilizing substitutions and determining whether the NANOBODIES    still bind RANK-L and whether the favourable properties of    NANOBODIES are obtained or improved (i.e. compared to the original    V_(H) or V_(HH) domain). A preferred stabilized NANOBODY is depicted    in SEQ ID NO: 756) wherein the DS motif in CDR2 is replaced with ES    resulting in the following CDR2: SITGSGGSTYYAESVKG (SEQ ID NO: 758).

As will also be clear from the disclosure herein, it is also within thescope of the invention to use parts or fragments, or combinations of twoor more parts or fragments, of the NANOBODIES of the invention asdefined herein, and in particular parts or fragments of the NANOBODIESof SEQ ID NO's: 560-621. Thus, according to one aspect of the invention,the term “NANOBODY of the invention” in its broadest sense also coverssuch parts or fragments.

Generally, such parts or fragments of the NANOBODIES of the invention(including analogs thereof) have amino acid sequences in which, comparedto the amino acid sequence of the corresponding full length NANOBODY ofthe invention (or analog thereof), one or more of the amino acidresidues at the N-terminal end, one or more amino acid residues at theC-terminal end, one or more contiguous internal amino acid residues, orany combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to RANK-Lwith an affinity (suitably measured and/or expressed as a K_(D)-value(actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rateand/or a k_(off)-rate, or alternatively as an IC₅₀ value, as furtherdescribed herein) that is as defined herein for the NANOBODIES of theinvention.

Any part or fragment is preferably such that it comprises at least 10contiguous amino acid residues, preferably at least 20 contiguous aminoacid residues, more preferably at least 30 contiguous amino acidresidues, such as at least 40 contiguous amino acid residues, of theamino acid sequence of the corresponding full length NANOBODY of theinvention.

Also, any part or fragment is such preferably that it comprises at leastone of CDR1, CDR2 and/or CDR3 or at least part thereof (and inparticular at least CDR3 or at least part thereof). More preferably, anypart or fragment is such that it comprises at least one of the CDR's(and preferably at least CDR3 or part thereof) and at least one otherCDR (i.e. CDR1 or CDR2) or at least part thereof, preferably connectedby suitable framework sequence(s) or at least part thereof. Morepreferably, any part or fragment is such that it comprises at least oneof the CDR's (and preferably at least CDR3 or part thereof) and at leastpart of the two remaining CDR's, again preferably connected by suitableframework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limiting aspect,such a part or fragment comprises at least CDR3, such as FR3, CDR3 andFR4 of the corresponding full length NANOBODY of the invention, i.e. asfor example described in the International application WO 03/050531(Lasters et al.).

As already mentioned above, it is also possible to combine two or moreof such parts or fragments (i.e. from the same or different NANOBODIESof the invention), i.e. to provide an analog (as defined herein) and/orto provide further parts or fragments (as defined herein) of a NANOBODYof the invention. It is for example also possible to combine one or moreparts or fragments of a NANOBODY of the invention with one or more partsor fragments of a human V_(H) domain.

According to one preferred aspect, the parts or fragments have a degreeof sequence identity of at least 50%, preferably at least 60%, morepreferably at least 70%, even more preferably at least 80%, such as atleast 90%, 95% or 99% or more with one of the NANOBODIES of SEQ ID NOs:560-621.

The parts and fragments, and nucleic acid sequences encoding the same,can be provided and optionally combined in any manner known per se. Forexample, such parts or fragments can be obtained by inserting a stopcodon in a nucleic acid that encodes a full-sized NANOBODY of theinvention, and then expressing the nucleic acid thus obtained in amanner known per se (e.g. as described herein). Alternatively, nucleicacids encoding such parts or fragments can be obtained by suitablyrestricting a nucleic acid that encodes a full-sized NANOBODY of theinvention or by synthesizing such a nucleic acid in a manner known perse. Parts or fragments may also be provided using techniques for peptidesynthesis known per se.

The invention in its broadest sense also comprises derivatives of theNANOBODIES of the invention. Such derivatives can generally be obtainedby modification, and in particular by chemical and/or biological (e.g.enzymatical) modification, of the NANOBODIES of the invention and/or ofone or more of the amino acid residues that form the NANOBODIES of theinvention.

Examples of such modifications, as well as examples of amino acidresidues within the NANOBODY sequence that can be modified in such amanner (i.e. either on the protein backbone but preferably on a sidechain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the NANOBODY of theinvention, and in particular of one or more functional groups, residuesor moieties that confer one or more desired properties orfunctionalities to the NANOBODY of the invention. Example of suchfunctional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that increase the half-life, the solubility and/or theabsorption of the NANOBODY of the invention, that reduce theimmunogenicity and/or the toxicity of the NANOBODY of the invention,that eliminate or attenuate any undesirable side effects of the NANOBODYof the invention, and/or that confer other advantageous properties toand/or reduce the undesired properties of the NANOBODIES and/orpolypeptides of the invention; or any combination of two or more of theforegoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and single domain antibodies), for whichreference is for example made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may for example be linked directly (for example covalently) to aNANOBODY of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or reducing the immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman,Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug.Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylationof proteins are also commercially available, for example from NektarTherapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering, 16,10, 761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in a NANOBODY of theinvention, a NANOBODY of the invention may be modified so as to suitablyintroduce one or more cysteine residues for attachment of PEG, or anamino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of a NANOBODYof the invention, all using techniques of protein engineering known perse to the skilled person.

Preferably, for the NANOBODIES and proteins of the invention, a PEG isused with a molecular weight of more than 5000, such as more than 10,000and less than 200,000, such as less than 100,000; for example in therange of 20,000-80,000.

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the NANOBODY or polypeptide of the invention.

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the labelled NANOBODY. Suitable labelsand techniques for attaching, using and detecting them will be clear tothe skilled person, and for example include, but are not limited to,fluorescent labels (such as fluorescein, isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, andfluorescamine and fluorescent metals such as ¹⁵²Eu or others metals fromthe lanthanide series), phosphorescent labels, chemiluminescent labelsor bioluminescent labels (such as luminal, isoluminol, theromaticacridinium ester, imidazole, acridinium salts, oxalate ester, dioxetaneor GFP and its analogs), radio-isotopes (such as ³H, ¹²⁵I, ³²P, ³⁵S,¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, and ⁷⁵Se), metals, metal chelates ormetallic cations (for example metallic cations such as ^(99m)Tc, ¹²³I,¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, and ⁶⁸Ga or other metals or metalliccations that are particularly suited for use in in vivo, in vitro or insitu diagnosis and imaging, such as (¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and⁵⁶Fe), as well as chromophores and enzymes (such as malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinavidin peroxidase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholine esterase). Other suitable labels will beclear to the skilled person, and for example include moieties that canbe detected using NMR or ESR spectroscopy.

Such labelled NANOBODIES and polypeptides of the invention may forexample be used for in vitro, in vivo or in situ assays (includingimmunoassays known per se such as ELISA, RIA, EIA and other “sandwichassays”, etc.) as well as in vivo diagnostic and imaging purposes,depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groupsfor example include, without limitation, diethyl-enetriaminepentaaceticacid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functionalgroup that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair. Such a functional group may be usedto link the NANOBODY of the invention to another protein, polypeptide orchemical compound that is bound to the other half of the binding pair,i.e. through formation of the binding pair. For example, a NANOBODY ofthe invention may be conjugated to biotin, and linked to anotherprotein, polypeptide, compound or carrier conjugated to avidin orstreptavidin. For example, such a conjugated NANOBODY may be used as areporter, for example in a diagnostic system where a detectablesignal-producing agent is conjugated to avidin or streptavidin. Suchbinding pairs may for example also be used to bind the NANOBODY of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example are the liposomal formulationsdescribed by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257(2000). Such binding pairs may also be used to link a therapeuticallyactive agent to the NANOBODY of the invention.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNANOBODIES of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation such acell, the NANOBODIES of the invention may also be linked to a toxin orto a toxic residue or moiety. Examples of toxic moieties, compounds orresidues which can be linked to a NANOBODY of the invention toprovide—for example—a cytotoxic compound will be clear to the skilledperson and can for example be found in the prior art cited above and/orin the further description herein. One example is the so-called ADEPT™technology described in WO 03/055527.

Other potential chemical and enzymatical modifications will be clear tothe skilled person. Such modifications may also be introduced forresearch purposes (e.g. to study function-activity relationships).Reference is for example made to Lundblad and Bradshaw, Biotechnol.Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to RANK-L with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein for the NANOBODIES of the invention.

As mentioned above, the invention also relates to proteins orpolypeptides that essentially consist of or comprise at least oneNANOBODY of the invention. By “essentially consist of” is meant that theamino acid sequence of the polypeptide of the invention either isexactly the same as the amino acid sequence of a NANOBODY of theinvention or corresponds to the amino acid sequence of a NANOBODY of theinvention which has a limited number of amino acid residues, such as1-20 amino acid residues, for example 1-10 amino acid residues andpreferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 aminoacid residues, added at the amino terminal end, at the carboxy terminalend, or at both the amino terminal end and the carboxy terminal end ofthe amino acid sequence of the NANOBODY.

Said amino acid residues may or may not change, alter or otherwiseinfluence the (biological) properties of the NANOBODY and may or may notadd further functionality to the NANOBODY. For example, such amino acidresidues:

-   -   can comprise an N-terminal Met residue, for example as result of        expression in a heterologous host cell or host organism.    -   may form a signal sequence or leader sequence that directs        secretion of the NANOBODY from a host cell upon synthesis.        Suitable secretory leader peptides will be clear to the skilled        person, and may be as further described herein. Usually, such a        leader sequence will be linked to the N-terminus of the        NANOBODY, although the invention in its broadest sense is not        limited thereto;    -   may form a sequence or signal that allows the NANOBODY to be        directed towards and/or to penetrate or enter into specific        organs, tissues, cells, or parts or compartments of cells,        and/or that allows the NANOBODY to penetrate or cross a        biological barrier such as a cell membrane, a cell layer such as        a layer of epithelial cells, a tumor including solid tumors, or        the blood-brain-barrier. Examples of such amino acid sequences        will be clear to the skilled person. Some non-limiting examples        are the small peptide vectors (“Pep-trans vectors”) described in        WO 03/026700 and in Temsamani et al., Expert Opin. Biol. Ther.,        1, 773 (2001); Temsamani and Vidal, Drug Discov. Today, 9,        1012 (004) and Rousselle, J. Pharmacol. Exp. Ther., 296, 124-131        (2001), and the membrane translocator sequence described by Zhao        et al., Apoptosis, 8, 631-637 (2003). C-terminal and N-terminal        amino acid sequences for intracellular targeting of antibody        fragments are for example described by Cardinale et al.,        Methods, 34, 171 (2004). Other suitable techniques for        intracellular targeting involve the expression and/or use of        so-called “intrabodies” comprising a NANOBODY of the invention,        as mentioned below;    -   may form a “tag”, for example an amino acid sequence or residue        that allows or facilitates the purification of the NANOBODY, for        example using affinity techniques directed against said sequence        or residue. Thereafter, said sequence or residue may be removed        (e.g. by chemical or enzymatical cleavage) to provide the        NANOBODY sequence (for this purpose, the tag may optionally be        linked to the NANOBODY sequence via a cleavable linker sequence        or contain a cleavable motif). Some preferred, but non-limiting        examples of such residues are multiple histidine residues,        glutathione residues and a myc-tag (see for example SEQ ID NO:31        of WO 06/12282).    -   may be one or more amino acid residues that have been        functionalized and/or that can serve as a site for attachment of        functional groups. Suitable amino acid residues and functional        groups will be clear to the skilled person and include, but are        not limited to, the amino acid residues and functional groups        mentioned herein for the derivatives of the NANOBODIES of the        invention.

According to another aspect, a polypeptide of the invention comprises aNANOBODY of the invention, which is fused at its amino terminal end, atits carboxy terminal end, or both at its amino terminal end and at itscarboxy terminal end to at least one further amino acid sequence, i.e.so as to provide a fusion protein comprising said NANOBODY of theinvention and the one or more further amino acid sequences. Such afusion will also be referred to herein as a “NANOBODY fusion”.

The one or more further amino acid sequence may be any suitable and/ordesired amino acid sequences. The further amino acid sequences may ormay not change, alter or otherwise influence the (biological) propertiesof the NANOBODY, and may or may not add further functionality to theNANOBODY or the polypeptide of the invention. Preferably, the furtheramino acid sequence is such that it confers one or more desiredproperties or functionalities to the NANOBODY or the polypeptide of theinvention.

For example, the further amino acid sequence may also provide a secondbinding site, which binding site may be directed against any desiredprotein, polypeptide, antigen, antigenic determinant or epitope(including but not limited to the same protein, polypeptide, antigen,antigenic determinant or epitope against which the NANOBODY of theinvention is directed, or a different protein, polypeptide, antigen,antigenic determinant or epitope).

Example of such amino acid sequences will be clear to the skilledperson, and may generally comprise all amino acid sequences that areused in peptide fusions based on conventional antibodies and fragmentsthereof (including but not limited to ScFv's and single domainantibodies). Reference is for example made to the review by Holliger andHudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

For example, such an amino acid sequence may be an amino acid sequencethat increases the half-life, the solubility, or the absorption, reducesthe immunogenicity or the toxicity, eliminates or attenuates undesirableside effects, and/or confers other advantageous properties to and/orreduces the undesired properties of the polypeptides of the invention,compared to the NANOBODY of the invention per se. Some non-limitingexamples of such amino acid sequences are serum proteins, such as humanserum albumin (see for example WO 00/27435) or haptenic molecules (forexample haptens that are recognized by circulating antibodies, see forexample WO 98/22141).

In particular, it has been described in the art that linking fragmentsof immunoglobulins (such as V_(H) domains) to serum albumin or tofragments thereof can be used to increase the half-life. Reference isfor made to WO 00/27435 and WO 01/077137). According to the invention,the NANOBODY of the invention is preferably either directly linked toserum albumin (or to a suitable fragment thereof) or via a suitablelinker, and in particular via a suitable peptide linked so that thepolypeptide of the invention can be expressed as a genetic fusion(protein). According to one specific aspect, the NANOBODY of theinvention may be linked to a fragment of serum albumin that at leastcomprises the domain III of serum albumin or part thereof. Reference isfor example made to WO 07/112940 of Ablynx N.V.

Alternatively, the further amino acid sequence may provide a secondbinding site or binding unit that is directed against a serum protein(such as, for example, human serum albumin or another serum protein suchas IgG), so as to provide increased half-life in serum. Such amino acidsequences for example include the NANOBODIES described below, as well asthe small peptides and binding proteins described in WO 91/01743, WO01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO04/003019. Reference is also made to Harmsen et al., Vaccine, 23 (41);4926-42, 2005, as well as to EP 0 368 684, as well as to the followingthe U.S. provisional applications 60/843,349 (see alsoPCT/EP2007/059475), 60/850,774 (see also PCT/EP2007/060849), 60/850,775(see also PCT/EP2007/060850) by Ablynx N.V. mentioned herein and USprovisional application of Ablynx N.V. entitled “Peptides capable ofbinding to serum proteins” filed on Dec. 5, 2006 (see alsoPCT/EP2007/063348).

Such amino acid sequences may in particular be directed against serumalbumin (and more in particular human serum albumin) and/or against IgG(and more in particular human IgG). For example, such amino acidsequences may be amino acid sequences that are directed against (human)serum albumin and amino acid sequences that can bind to amino acidresidues on (human) serum albumin that are not involved in binding ofserum albumin to FcRn (see for example WO 06/0122787) and/or amino acidsequences that are capable of binding to amino acid residues on serumalbumin that do not form part of domain III of serum albumin (see againfor example WO 06/0122787); amino acid sequences that have or canprovide an increased half-life (see for example WO 08/028977 by AblynxN.V.); amino acid sequences against human serum albumin that arecross-reactive with serum albumin from at least one species of mammal,and in particular with at least one species of primate (such as, withoutlimitation, monkeys from the genus Macaca (such as, and in particular,cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macacamulatta)) and baboon (Papio ursinus), reference is again made to theU.S. provisional application 60/843,349 and PCT/EP2007/059475); aminoacid sequences that can bind to serum albumin in a pH independent manner(see for example the U.S. provisional application 60/850,774 by AblynxN.V. entitled “Amino acid sequences that bind to serum proteins in amanner that is essentially independent of the pH, compounds comprisingthe same, and uses thereof”, filed on Oct. 11, 2006; see also andPCT/EP2007/059475) and/or amino acid sequences that are conditionalbinders (see for example the U.S. provisional application 60/850,775 byAblynx N.V. entitled “Amino acid sequences that bind to a desiredmolecule in a conditional manner”, filed on Oct. 11, 2006; see alsoPCT/EP2007/060850).

According to another aspect, the one or more further amino acidsequences may comprise one or more parts, fragments or domains ofconventional 4-chain antibodies (and in particular human antibodies)and/or of heavy chain antibodies. For example, although usually lesspreferred, a NANOBODY of the invention may be linked to a conventional(preferably human) V_(H) or V_(L) domain or to a natural or syntheticanalog of a V_(H) or V_(L) domain, again optionally via a linkersequence (including but not limited to other (single) domain antibodies,such as the dAb's described by Ward et al.).

The at least one NANOBODY may also be linked to one or more (preferablyhuman) C_(H)1, C_(H)2 and/or C_(H)3 domains, optionally via a linkersequence. For instance, a NANOBODY linked to a suitable C_(H)1 domaincould for example be used—together with suitable light chains—togenerate antibody fragments/structures analogous to conventional Fabfragments or F(ab′)₂ fragments, but in which one or (in case of anF(ab′)₂ fragment) one or both of the conventional V_(H) domains havebeen replaced by a NANOBODY of the invention. Also, two NANOBODIES couldbe linked to a C_(H)3 domain (optionally via a linker) to provide aconstruct with increased half-life in vivo.

According to one specific aspect of a polypeptide of the invention, oneor more NANOBODIES of the invention may be linked (optionally via asuitable linker or hinge region) to one or more constant domains (forexample, 2 or 3 constant domains that can be used as part of/to form anFc portion), to an Fc portion and/or to one or more antibody parts,fragments or domains that confer one or more effector functions to thepolypeptide of the invention and/or may confer the ability to bind toone or more Fc receptors. For example, for this purpose, and withoutbeing limited thereto, the one or more further amino acid sequences maycomprise one or more C_(H)2 and/or C_(H)3 domains of an antibody, suchas from a heavy chain antibody (as described herein) and more preferablyfrom a conventional human 4-chain antibody; and/or may form (part of)and Fc region, for example from IgG (e.g. from IgG1, IgG2, IgG3 orIgG4), from IgE or from another human Ig such as IgA, IgD or IgM. Forexample, WO 94/04678 describes heavy chain antibodies comprising aCamelid V_(HH) domain or a humanized derivative thereof (i.e. aNANOBODY), in which the Camelidae C_(H)2 and/or C_(H)3 domain have beenreplaced by human C_(H)2 and C_(H)3 domains, so as to provide animmunoglobulin that consists of 2 heavy chains each comprising aNANOBODY and human C_(H)2 and C_(H)3 domains (but no C_(H)1 domain),which immunoglobulin has the effector function provided by the C_(H)2and C_(H)3 domains and which immunoglobulin can function without thepresence of any light chains. Other amino acid sequences that can besuitably linked to the NANOBODIES of the invention so as to provide aneffector function will be clear to the skilled person, and may be chosenon the basis of the desired effector function(s). Reference is forexample made to WO 04/058820, WO 99/42077, WO 02/056910 and WO05/017148, as well as the review by Holliger and Hudson, supra and tothe non-prepublished US provisional application by Ablynx N.V. entitled“Constructs comprising single variable domains and an Fc portion derivedfrom IgE” which has a filing date of Dec. 4, 2007. Coupling of aNANOBODY of the invention to an Fc portion may also lead to an increasedhalf-life, compared to the corresponding NANOBODY of the invention. Forsome applications, the use of an Fc portion and/or of constant domains(i.e. C_(H)2 and/or C_(H)3 domains) that confer increased half-lifewithout any biologically significant effector function may also besuitable or even preferred. Other suitable constructs comprising one ormore NANOBODIES and one or more constant domains with increasedhalf-life in vivo will be clear to the skilled person, and may forexample comprise two NANOBODIES linked to a C_(H)3 domain, optionallyvia a linker sequence. Generally, any fusion protein or derivatives withincreased half-life will preferably have a molecular weight of more than50 kD, the cut-off value for renal absorption.

In another one specific, but non-limiting, aspect, in order to form apolypeptide of the invention, one or more amino acid sequences of theinvention may be linked (optionally via a suitable linker or hingeregion) to naturally occurring, synthetic or semisynthetic constantdomains (or analogs, variants, mutants, parts or fragments thereof) thathave a reduced (or essentially no) tendency to self-associate intodimers (i.e. compared to constant domains that naturally occur inconventional 4-chain antibodies). Such monomeric (i.e. notself-associating) Fc chain variants, or fragments thereof, will be clearto the skilled person. For example, Helm et al., J Biol Chem 1996 2717494, describe monomeric FcE chain variants that can be used in thepolypeptide chains of the invention.

Also, such monomeric Fc chain variants are preferably such that they arestill capable of binding to the complement or the relevant Fcreceptor(s) (depending on the Fc portion from which they are derived),and/or such that they still have some or all of the effector functionsof the Fc portion from which they are derived (or at a reduced levelstill suitable for the intended use). Alternatively, in such apolypeptide chain of the invention, the monomeric Fc chain may be usedto confer increased half-life upon the polypeptide chain, in which casethe monomeric Fc chain may also have no or essentially no effectorfunctions.

Bivalent/multivalent, bispecific/multispecific orbiparatopic/multiparatopic polypeptides of the invention may also belinked to Fc portions, in order to provide polypeptide constructs of thetype that is described in the non-prepublished US provisionalapplication U.S. 61/005,331 entitled “immunoglobulin constructs” filedon Dec. 4, 2007.

The further amino acid sequences may also form a signal sequence orleader sequence that directs secretion of the NANOBODY or thepolypeptide of the invention from a host cell upon synthesis (forexample to provide a pre-, pro- or prepro- form of the polypeptide ofthe invention, depending on the host cell used to express thepolypeptide of the invention).

The further amino acid sequence may also form a sequence or signal thatallows the NANOBODY or polypeptide of the invention to be directedtowards and/or to penetrate or enter into specific organs, tissues,cells, or parts or compartments of cells, and/or that allows theNANOBODY or polypeptide of the invention to penetrate or cross abiological barrier such as a cell membrane, a cell layer such as a layerof epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Suitable examples of such amino acid sequences willbe clear to the skilled person, and for example include, but are notlimited to, the “Peptrans” vectors mentioned above, the sequencesdescribed by Cardinale et al. and the amino acid sequences and antibodyfragments known per se that can be used to express or produce theNANOBODIES and polypeptides of the invention as so-called “intrabodies”,for example as described in WO 94/02610, WO 95/22618, U.S. Pat. No.7,004,940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and inCattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Developmentand Applications. Landes and Springer-Verlag; and in Kontermann, Methods34, (2004), 163-170, and the further references described therein.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNANOBODIES of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation of such acell, the NANOBODIES of the invention may also be linked to a(cyto)toxic protein or polypeptide. Examples of such toxic proteins andpolypeptides which can be linked to a NANOBODY of the invention toprovide—for example—a cytotoxic polypeptide of the invention will beclear to the skilled person and can for example be found in the priorart cited above and/or in the further description herein. One example isthe so-called ADEPT™ technology described in WO 03/055527.

According to one preferred, but non-limiting aspect, said one or morefurther amino acid sequences comprise at least one further NANOBODY, soas to provide a polypeptide of the invention that comprises at leasttwo, such as three, four, five or more NANOBODIES, in which saidNANOBODIES may optionally be linked via one or more linker sequences (asdefined herein). Polypeptides of the invention that comprise two or moreNANOBODIES, of which at least one is a NANOBODY of the invention, willalso be referred to herein as “multivalent” polypeptides of theinvention, and the NANOBODIES present in such polypeptides will also bereferred to herein as being in a “multivalent format”. For example a“bivalent” polypeptide of the invention comprises two NANOBODIES,optionally linked via a linker sequence, whereas a “trivalent”polypeptide of the invention comprises three NANOBODIES, optionallylinked via two linker sequences; etc.; in which at least one of theNANOBODIES present in the polypeptide, and up to all of the NANOBODIESpresent in the polypeptide, is/are a NANOBODY of the invention.

In a multivalent polypeptide of the invention, the two or moreNANOBODIES may be the same or different, and may be directed against thesame antigen or antigenic determinant (for example against the samepart(s) or epitope(s) or against different parts or epitopes) or mayalternatively be directed against different antigens or antigenicdeterminants; or any suitable combination thereof. For example, abivalent polypeptide of the invention may comprise (a) two identicalNANOBODIES; (b) a first NANOBODY directed against a first antigenicdeterminant of a protein or antigen and a second NANOBODY directedagainst the same antigenic determinant of said protein or antigen whichis different from the first NANOBODY; (c) a first NANOBODY directedagainst a first antigenic determinant of a protein or antigen and asecond NANOBODY directed against another antigenic determinant of saidprotein or antigen; or (d) a first NANOBODY directed against a firstprotein or antigen and a second NANOBODY directed against a secondprotein or antigen (i.e. different from said first antigen). Similarly,a trivalent polypeptide of the invention may, for example and withoutbeing limited thereto. comprise (a) three identical NANOBODIES; (b) twoidentical NANOBODY against a first antigenic determinant of an antigenand a third NANOBODY directed against a different antigenic determinantof the same antigen; (c) two identical NANOBODY against a firstantigenic determinant of an antigen and a third NANOBODY directedagainst a second antigen different from said first antigen; (d) a firstNANOBODY directed against a first antigenic determinant of a firstantigen, a second NANOBODY directed against a second antigenicdeterminant of said first antigen and a third NANOBODY directed againsta second antigen different from said first antigen; or (e) a firstNANOBODY directed against a first antigen, a second NANOBODY directedagainst a second antigen different from said first antigen, and a thirdNANOBODY directed against a third antigen different from said first andsecond antigen.

Polypeptides of the invention that contain at least two NANOBODIES, inwhich at least one NANOBODY is directed against a first antigen (i.e.against RANK-L) and at least one NANOBODY is directed against a secondantigen (i.e. different from RANK-L), will also be referred to as“multispecific” polypeptides of the invention, and the NANOBODIESpresent in such polypeptides will also be referred to herein as being ina “multispecific format”. Thus, for example, a “bispecific” polypeptideof the invention is a polypeptide that comprises at least one NANOBODYdirected against a first antigen (i.e. RANK-L) and at least one furtherNANOBODY directed against a second antigen (i.e. different from RANK-L),whereas a “trispecific” polypeptide of the invention is a polypeptidethat comprises at least one NANOBODY directed against a first antigen(i.e. RANK-L), at least one further NANOBODY directed against a secondantigen (i.e. different from RANK-L) and at least one further NANOBODYdirected against a third antigen (i.e. different from both RANK-L, andthe second antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first NANOBODY directed against RANK-L, and asecond NANOBODY directed against a second antigen, in which said firstand second NANOBODY may optionally be linked via a linker sequence (asdefined herein); whereas a trispecific polypeptide of the invention inits simplest form is a trivalent polypeptide of the invention (asdefined herein), comprising a first NANOBODY directed against RANK-L, asecond NANOBODY directed against a second antigen and a third NANOBODYdirected against a third antigen, in which said first, second and thirdNANOBODY may optionally be linked via one or more, and in particular oneand more, in particular two, linker sequences.

However, as will be clear from the description hereinabove, theinvention is not limited thereto, in the sense that a multispecificpolypeptide of the invention may comprise at least one NANOBODY againstRANK-L, and any number of NANOBODIES directed against one or moreantigens different from RANK-L.

Furthermore, although it is encompassed within the scope of theinvention that the specific order or arrangement of the variousNANOBODIES in the polypeptides of the invention may have some influenceon the properties of the final polypeptide of the invention (includingbut not limited to the affinity, specificity or avidity for RANK-L, oragainst the one or more other antigens), said order or arrangement isusually not critical and may be suitably chosen by the skilled person,optionally after some limited routine experiments based on thedisclosure herein. Thus, when reference is made to a specificmultivalent or multispecific polypeptide of the invention, it should benoted that this encompasses any order or arrangements of the relevantNANOBODIES, unless explicitly indicated otherwise.

Finally, it is also within the scope of the invention that thepolypeptides of the invention contain two or more NANOBODIES and one ormore further amino acid sequences (as mentioned herein).

For multivalent and multispecific polypeptides containing one or moreV_(HH) domains and their preparation, reference is also made to Conrathet al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans,Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to forexample WO 96/34103 and WO 99/23221. Some other examples of somespecific multispecific and/or multivalent polypeptide of the inventioncan be found in the applications by Ablynx N.V. referred to herein.

One preferred, but non-limiting example of a multispecific polypeptideof the invention comprises at least one NANOBODY of the invention and atleast one NANOBODY that provides for an increased half-life. SuchNANOBODIES may for example be NANOBODIES that are directed against aserum protein, and in particular a human serum protein, such as humanserum albumin, thyroxine-binding protein, (human) transferrin,fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or against one ofthe serum proteins listed in WO 04/003019. Of these, NANOBODIES that canbind to serum albumin (and in particular human serum albumin) or to IgG(and in particular human IgG, see for example NANOBODY VH-1 described inthe review by Muyldermans, supra) are particularly preferred (althoughfor example, for experiments in mice or primates, NANOBODIES against orcross-reactive with mouse serum albumin (MSA) or serum albumin from saidprimate, respectively, can be used. However, for pharmaceutical use,NANOBODIES against human serum albumin or human IgG will usually bepreferred). NANOBODIES that provide for increased half-life and that canbe used in the polypeptides of the invention include the NANOBODIESdirected against serum albumin that are described in WO 04/041865, in WO06/122787 and in the further patent applications by Ablynx N.V., such asthose mentioned above.

For example, the some preferred NANOBODIES that provide for increasedhalf-life for use in the present invention include NANOBODIES that canbind to amino acid residues on (human) serum albumin that are notinvolved in binding of serum albumin to FcRn (see for example WO06/0122787); NANOBODIES that are capable of binding to amino acidresidues on serum albumin that do not form part of domain III of serumalbumin (see for example WO 06/0122787); NANOBODIES that have or canprovide an increased half-life (see for example the U.S. provisionalapplication 60/843,349 by Ablynx N.V mentioned herein; see alsoPCT/EP2007/059475); NANOBODIES against human serum albumin that arecross-reactive with serum albumin from at least one species of mammal,and in particular with at least one species of primate (such as, withoutlimitation, monkeys from the genus Macaca (such as, and in particular,cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macacamulatta)) and baboon (Papio ursinus)) (see for example the U.S.provisional application 60/843,349 by Ablynx N.V; see alsoPCT/EP2007/059475); NANOBODIES that can bind to serum albumin in a pHindependent manner (see for example the U.S. provisional application60/850,774 by Ablynx N.V.; see also PCT/EP2007/060849) and/or NANOBODIESthat are conditional binders (see for example the U.S. provisionalapplication 60/850,775 by Ablynx N.V.; see also PCT/EP2007/060850).

Some particularly preferred NANOBODIES that provide for increasedhalf-life and that can be used in the polypeptides of the inventioninclude the NANOBODIES ALB-1 to ALB-10 disclosed in WO 06/122787 (seeTables II and III) of which ALB-8 (SEQ ID NO: 62 in WO 06/122787) isparticularly preferred.

Some preferred, but non-limiting examples of polypeptides of theinvention that comprise at least one NANOBODY of the invention and atleast one NANOBODY that provides for increased half-life are given inSEQ ID NO's 694-729 and 759-760.

According to a specific, but non-limiting aspect of the invention, thepolypeptides of the invention contain, besides the one or moreNANOBODIES of the invention, at least one NANOBODY against human serumalbumin.

Generally, any polypeptides of the invention with increased half-lifethat contain one or more NANOBODIES of the invention, and anyderivatives of NANOBODIES of the invention or of such polypeptides thathave an increased half-life, preferably have a half-life that is atleast 1.5 times, preferably at least 2 times, such as at least 5 times,for example at least 10 times or more than 20 times, greater than thehalf-life of the corresponding NANOBODY of the invention per se. Forexample, such a derivative or polypeptides with increased half-life mayhave a half-life that is increased with more than 1 hours, preferablymore than 2 hours, more preferably more than 6 hours, such as more than12 hours, or even more than 24, 48 or 72 hours, compared to thecorresponding NANOBODY of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchderivatives or polypeptides may exhibit a serum half-life in human of atleast about 12 hours, preferably at least 24 hours, more preferably atleast 48 hours, even more preferably at least 72 hours or more. Forexample, such derivatives or polypeptides may have a half-life of atleast 5 days (such as about 5 to 10 days), preferably at least 9 days(such as about 9 to 14 days), more preferably at least about 10 days(such as about 10 to 15 days), or at least about 11 days (such as about11 to 16 days), more preferably at least about 12 days (such as about 12to 18 days or more), or more than 14 days (such as about 14 to 19 days).

According to one aspect of the invention the polypeptides are capable ofbinding to one or more molecules which can increase the half-life of thepolypeptide in vivo.

The polypeptides of the invention are stabilised in vivo and theirhalf-life increased by binding to molecules which resist degradationand/or clearance or sequestration. Typically, such molecules arenaturally occurring proteins which themselves have a long half-life invivo.

Another preferred, but non-limiting example of a multispecificpolypeptide of the invention comprises at least one NANOBODY of theinvention and at least one NANOBODY that directs the polypeptide of theinvention towards, and/or that allows the polypeptide of the inventionto penetrate or to enter into specific organs, tissues, cells, or partsor compartments of cells, and/or that allows the NANOBODY to penetrateor cross a biological barrier such as a cell membrane, a cell layer suchas a layer of epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Examples of such NANOBODIES include NANOBODIES thatare directed towards specific cell-surface proteins, markers or epitopesof the desired organ, tissue or cell (for example cell-surface markersassociated with tumor cells), and the single-domain brain targetingantibody fragments described in WO 02/057445 and WO 06/040153, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

In the polypeptides of the invention, the one or more NANOBODIES and theone or more polypeptides may be directly linked to each other (as forexample described in WO 99/23221) and/or may be linked to each other viaone or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecificpolypeptides will be clear to the skilled person, and may generally beany linker or spacer used in the art to link amino acid sequences.Preferably, said linker or spacer is suitable for use in constructingproteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers thatare used in the art to link antibody fragments or antibody domains.These include the linkers mentioned in the general background art citedabove, as well as for example linkers that are used in the art toconstruct diabodies or ScFv fragments (in this respect, however, itsshould be noted that, whereas in diabodies and in ScFv fragments, thelinker sequence used should have a length, a degree of flexibility andother properties that allow the pertinent V_(H) and V_(L) domains tocome together to form the complete antigen-binding site, there is noparticular limitation on the length or the flexibility of the linkerused in the polypeptide of the invention, since each NANOBODY by itselfforms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and inparticular amino acid sequences of between 1 and 50, preferably between1 and 30, such as between 1 and 10 amino acid residues. Some preferredexamples of such amino acid sequences include gly-ser linkers, forexample of the type (gly_(x)ser_(y))_(x), such as (for example(gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077 and the GS30,GS15, GS9 and GS7 linkers described in the applications by Ablynxmentioned herein (see for example WO 06/040153 and WO 06/122825), aswell as hinge-like regions, such as the hinge regions of naturallyoccurring heavy chain antibodies or similar sequences (such as describedin WO 94/04678).

Some other particularly preferred linkers are poly-alanine (such asAAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) andGS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, poly(ethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, thedegree of flexibility and/or other properties of the linker(s) used(although not critical, as it usually is for linkers used in ScFvfragments) may have some influence on the properties of the finalpolypeptide of the invention, including but not limited to the affinity,specificity or avidity for RANK-L, or for one or more of the otherantigens. Based on the disclosure herein, the skilled person will beable to determine the optimal linker(s) for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

For example, in multivalent polypeptides of the invention that compriseNANOBODIES directed against a multimeric antigen (such as a multimericreceptor or other protein), the length and flexibility of the linker arepreferably such that it allows each NANOBODY of the invention present inthe polypeptide to bind to the antigenic determinant on each of thesubunits of the multimer. Similarly, in a multispecific polypeptide ofthe invention that comprises NANOBODIES directed against two or moredifferent antigenic determinants on the same antigen (for exampleagainst different epitopes of an antigen and/or against differentsubunits of a multimeric receptor, channel or protein), the length andflexibility of the linker are preferably such that it allows eachNANOBODY to bind to its intended antigenic determinant Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linker(s) for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to thepolypeptides of the invention, and/or provide one or more sites for theformation of derivatives and/or for the attachment of functional groups(e.g. as described herein for the derivatives of the NANOBODIES of theinvention). For example, linkers containing one or more charged aminoacid residues (see Table A-2 above) can provide improved hydrophilicproperties, whereas linkers that form or contain small epitopes or tagscan be used for the purposes of detection, identification and/orpurification. Again, based on the disclosure herein, the skilled personwill be able to determine the optimal linkers for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

Finally, when two or more linkers are used in the polypeptides of theinvention, these linkers may be the same or different. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of theinvention will be a linear polypeptide. However, the invention in itsbroadest sense is not limited thererto. For example, when a polypeptideof the invention comprises three of more NANOBODIES, it is possible tolink them by use of a linker with three or more “arms”, which each “arm”being linked to a NANOBODY, so as to provide a “star-shaped” construct.It is also possible, although usually less preferred, to use circularconstructs.

The invention also comprises derivatives of the polypeptides of theinvention, which may be essentially analogous to the derivatives of theNANOBODIES of the invention, i.e. as described herein.

The invention also comprises proteins or polypeptides that “essentiallyconsist” of a polypeptide of the invention (in which the wording“essentially consist of” has essentially the same meaning as indicatedhereinabove).

According to one aspect of the invention, the polypeptide of theinvention is in essentially isolated from, as defined herein.

The amino acid sequences, NANOBODIES, polypeptides and nucleic acids ofthe invention can be prepared in a manner known per se, as will be clearto the skilled person from the further description herein. For example,the NANOBODIES and polypeptides of the invention can be prepared in anymanner known per se for the preparation of antibodies and in particularfor the preparation of antibody fragments (including but not limited to(single) domain antibodies and ScFv fragments). Some preferred, butnon-limiting methods for preparing the amino acid sequences, NANOBODIES,polypeptides and nucleic acids include the methods and techniquesdescribed herein.

As will be clear to the skilled person, one particularly useful methodfor preparing an amino acid sequence, NANOBODY and/or a polypeptide ofthe invention generally comprises the steps of:

-   i) the expression, in a suitable host cell or host organism (also    referred to herein as a “host of the invention”) or in another    suitable expression system of a nucleic acid that encodes said amino    acid sequence, NANOBODY or polypeptide of the invention (also    referred to herein as a “nucleic acid of the invention”), optionally    followed by:-   ii) isolating and/or purifying the amino acid sequence, NANOBODY or    polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of:

-   i) cultivating and/or maintaining a host of the invention under    conditions that are such that said host of the invention expresses    and/or produces at least one amino acid sequence, NANOBODY and/or    polypeptide of the invention; optionally followed by:-   ii) isolating and/or purifying the amino acid sequence, NANOBODY or    polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA, and is preferably in the form of double strandedDNA. For example, the nucleotide sequences of the invention may begenomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage thathas been specifically adapted for expression in the intended host cellor host organism).

According to one aspect of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the amino acidsequences for the polypeptides of the invention given herein, and/or canbe isolated from a suitable natural source. To provide analogs,nucleotide sequences encoding naturally occurring V_(HH) domains can forexample be subjected to site-directed mutagenesis, so at to provide anucleic acid of the invention encoding said analog. Also, as will beclear to the skilled person, to prepare a nucleic acid of the invention,also several nucleotide sequences, such as at least one nucleotidesequence encoding a NANOBODY and for example nucleic acids encoding oneor more linkers can be linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers. These and other techniques will beclear to the skilled person, and reference is again made to the standardhandbooks, such as Sambrook et al. and Ausubel et al., mentioned above,as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art. Such genetic constructs generally comprise at leastone nucleic acid of the invention that is optionally linked to one ormore elements of genetic constructs known per se, such as for exampleone or more suitable regulatory elements (such as a suitablepromoter(s), enhancer(s), terminator(s), etc.) and the further elementsof genetic constructs referred to herein. Such genetic constructscomprising at least one nucleic acid of the invention will also bereferred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable forindependent replication, maintenance and/or inheritance in the intendedhost organism. For instance, the genetic constructs of the invention maybe in the form of a vector, such as for example a plasmid, cosmid, YAC,a viral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g. in a suitable host cell, host organism and/orexpression system).

In a preferred but non-limiting aspect, a genetic construct of theinvention comprises

-   i) at least one nucleic acid of the invention; operably connected to-   ii) one or more regulatory elements, such as a promoter and    optionally a suitable terminator;    and optionally also-   iii) one or more further elements of genetic constructs known per    se;    in which the terms “regulatory element”, “promoter”, “terminator”    and “operably connected” have their usual meaning in the art (as    further described herein); and in which said “further elements”    present in the genetic constructs may for example be 3′- or 5′-UTR    sequences, leader sequences, selection markers, expression    markers/reporter genes, and/or elements that may facilitate or    increase (the efficiency of) transformation or integration. These    and other suitable elements for such genetic constructs will be    clear to the skilled person, and may for instance depend upon the    type of construct used, the intended host cell or host organism; the    manner in which the nucleotide sequences of the invention of    interest are to be expressed (e.g. via constitutive, transient or    inducible expression); and/or the transformation technique to be    used. For example, regulatory sequences, promoters and terminators    known per se for the expression and production of antibodies and    antibody fragments (including but not limited to (single) domain    antibodies and ScFv fragments) may be used in an essentially    analogous manner.

Preferably, in the genetic constructs of the invention, said at leastone nucleic acid of the invention and said regulatory elements, andoptionally said one or more further elements, are “operably linked” toeach other, by which is generally meant that they are in a functionalrelationship with each other. For instance, a promoter is considered“operably linked” to a coding sequence if said promoter is able toinitiate or otherwise control/regulate the transcription and/or theexpression of a coding sequence (in which said coding sequence should beunderstood as being “under the control of” said promoter). Generally,when two nucleotide sequences are operably linked, they will be in thesame orientation and usually also in the same reading frame. They willusually also be essentially contiguous, although this may also not berequired.

Preferably, the regulatory and further elements of the geneticconstructs of the invention are such that they are capable of providingtheir intended biological function in the intended host cell or hostorganism.

For instance, a promoter, enhancer or terminator should be “operable” inthe intended host cell or host organism, by which is meant that (forexample) said promoter should be capable of initiating or otherwisecontrolling/regulating the transcription and/or the expression of anucleotide sequence—e.g. a coding sequence—to which it is operablylinked (as defined herein).

Some particularly preferred promoters include, but are not limited to,promoters known per se for the expression in the host cells mentionedherein; and in particular promoters for the expression in the bacterialcells, such as those mentioned herein and/or those used in the Examples.

A selection marker should be such that it allows—i.e. under appropriateselection conditions—host cells and/or host organisms that have been(successfully) transformed with the nucleotide sequence of the inventionto be distinguished from host cells/organisms that have not been(successfully) transformed. Some preferred, but non-limiting examples ofsuch markers are genes that provide resistance against antibiotics (suchas kanamycin or ampicillin), genes that provide for temperatureresistance, or genes that allow the host cell or host organism to bemaintained in the absence of certain factors, compounds and/or (food)components in the medium that are essential for survival of thenon-transformed cells or organisms.

A leader sequence should be such that—in the intended host cell or hostorganism—it allows for the desired post-translational modificationsand/or such that it directs the transcribed mRNA to a desired part ororganelle of a cell. A leader sequence may also allow for secretion ofthe expression product from said cell. As such, the leader sequence maybe any pro-, pre-, or prepro-sequence operable in the host cell or hostorganism. Leader sequences may not be required for expression in abacterial cell. For example, leader sequences known per se for theexpression and production of antibodies and antibody fragments(including but not limited to single domain antibodies and ScFvfragments) may be used in an essentially analogous manner.

An expression marker or reporter gene should be such that—in the hostcell or host organism—it allows for detection of the expression of (agene or nucleotide sequence present on) the genetic construct. Anexpression marker may optionally also allow for the localisation of theexpressed product, e.g. in a specific part or organelle of a cell and/orin (a) specific cell(s), tissue(s), organ(s) or part(s) of amulticellular organism. Such reporter genes may also be expressed as aprotein fusion with the amino acid sequence of the invention. Somepreferred, but non-limiting examples include fluorescent proteins suchas GFP.

Some preferred, but non-limiting examples of suitable promoters,terminator and further elements include those that can be used for theexpression in the host cells mentioned herein; and in particular thosethat are suitable for expression in bacterial cells, such as thosementioned herein and/or those used in the Examples below. For some(further) non-limiting examples of the promoters, selection markers,leader sequences, expression markers and further elements that may bepresent/used in the genetic constructs of the invention—such asterminators, transcriptional and/or translational enhancers and/orintegration factors—reference is made to the general handbooks such asSambrook et al. and Ausubel et al. mentioned above, as well as to theexamples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, U.S. Pat.No. 7,207,410, U.S. Pat. No. 5,693,492 and EP 1 085 089. Other exampleswill be clear to the skilled person. Reference is also made to thegeneral background art cited above and the further references citedherein.

The genetic constructs of the invention may generally be provided bysuitably linking the nucleotide sequence(s) of the invention to the oneor more further elements described above, for example using thetechniques described in the general handbooks such as Sambrook et al.and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained byinserting a nucleotide sequence of the invention in a suitable(expression) vector known per se. Some preferred, but non-limitingexamples of suitable expression vectors are those used in the Examplesbelow, as well as those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the amino acid sequence, NANOBODY orpolypeptide of the invention. Suitable hosts or host cells will be clearto the skilled person, and may for example be any suitable fungal,prokaryotic or eukaryotic cell or cell line or any suitable fungal,prokaryotic or eukaryotic organism, for example:

-   -   a bacterial strain, including but not limited to gram-negative        strains such as strains of Escherichia coli; of Proteus, for        example of Proteus mirabilis; of Pseudomonas, for example of        Pseudomonas fluorescens; and gram-positive strains such as        strains of Bacillus, for example of Bacillus subtilis or of        Bacillus brevis; of Streptomyces, for example of Streptomyces        lividans; of Staphylococcus, for example of Staphylococcus        carnosus; and of Lactococcus, for example of Lactococcus lactis;    -   a fungal cell, including but not limited to cells from species        of Trichoderma, for example from Trichoderma reesei; of        Neurospora, for example from Neurospora crassa; of Sordaria, for        example from Sordaria macrospora; of Aspergillus, for example        from Aspergillus niger or from Aspergillus sojae; or from other        filamentous fungi;    -   a yeast cell, including but not limited to cells from species of        Saccharomyces, for example of Saccharomyces cerevisiae; of        Schizosaccharomyces, for example of Schizosaccharomyces pombe;        of Pichia, for example of Pichia pastoris or of Pichia        methanolica; of Hansenula, for example of Hansenula polymorpha;        of Kluyveromyces, for example of Kluyveromyces lactis; of        Arxula, for example of Arxula adeninivorans; of Yarrowia, for        example of Yarrowia lipolytica;    -   an amphibian cell or cell line, such as Xenopus oocytes;    -   an insect-derived cell or cell line, such as cells/cell lines        derived from lepidoptera, including but not limited to        Spodoptera SF9 and Sf21 cells or cells/cell lines derived from        Drosophila, such as Schneider and Kc cells;    -   a plant or plant cell, for example in tobacco plants; and/or    -   a mammalian cell or cell line, for example a cell or cell line        derived from a human, a cell or a cell line from mammals        including but not limited to CHO-cells, BHK-cells (for example        BHK-21 cells) and human cells or cell lines such as HeLa, COS        (for example COS-7) and PER.C6 cells;        as well as all other hosts or host cells known per se for the        expression and production of antibodies and antibody fragments        (including but not limited to (single) domain antibodies and        ScFv fragments), which will be clear to the skilled person.        Reference is also made to the general background art cited        hereinabove, as well as to for example WO 94/29457; WO 96/34103;        WO 99/42077; Frenken et al., (1998), supra; Riechmann and        Muyldermans, (1999), supra; van der Linden, (2000), supra;        Thomassen et al., (2002), supra; Joosten et al., (2003), supra;        Joosten et al., (2005), supra; and the further references cited        herein.

The amino acid sequences, NANOBODIES and polypeptides of the inventioncan also be introduced and expressed in one or more cells, tissues ororgans of a multicellular organism, for example for prophylactic and/ortherapeutic purposes (e.g. as a gene therapy). For this purpose, thenucleotide sequences of the invention may be introduced into the cellsor tissues in any suitable way, for example as such (e.g. usingliposomes) or after they have been inserted into a suitable gene therapyvector (for example derived from retroviruses such as adenovirus, orparvoviruses such as adeno-associated virus). As will also be clear tothe skilled person, such gene therapy may be performed in vivo and/or insitu in the body of a patient by administering a nucleic acid of theinvention or a suitable gene therapy vector encoding the same to thepatient or to specific cells or a specific tissue or organ of thepatient; or suitable cells (often taken from the body of the patient tobe treated, such as explanted lymphocytes, bone marrow aspirates ortissue biopsies) may be treated in vitro with a nucleotide sequence ofthe invention and then be suitably (re-)introduced into the body of thepatient. All this can be performed using gene therapy vectors,techniques and delivery systems which are well known to the skilledperson, and for example described in Culver, K. W., “Gene Therapy”,1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, N.Y);Giordano, Nature F Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79(1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ.Res. 77 (1995), 1077-1086; Onodera, Blood 91; (1998), 30-36; Verma, GeneTher. 5 (1998), 692-699; Nabel, Ann N.Y. Acad. Sci.: 811 (1997),289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang, NatureMedicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, U.S. Pat. No.5,580,859; U.S. Pat. No. 55,895,466; or Schaper, Current Opinion inBiotechnology 7 (1996), 635-640. For example, in situ expression of ScFvfragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and ofdiabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has beendescribed in the art.

For expression of the NANOBODIES in a cell, they may also be expressedas so-called “intrabodies”, as for example described in WO 94/02610, WO95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960; in Cattaneo, A. &Biocca, S. (1997) Intracellular Antibodies: Development andApplications. Landes and Springer-Verlag; and in Kontermann, Methods 34,(2004), 163-170.

The amino acid sequences, NANOBODIES and polypeptides of the inventioncan for example also be produced in the milk of transgenic mammals, forexample in the milk of rabbits, cows, goats or sheep (see for exampleU.S. Pat. No. 6,741,957, U.S. Pat. No. 6,304,489 and U.S. Pat. No.6,849,992 for general techniques for introducing transgenes intomammals), in plants or parts of plants including but not limited totheir leaves, flowers, fruits, seed, roots or tubers (for example intobacco, maize, soybean or alfalfa) or in for example pupae of thesilkworm Bombix mori.

Furthermore, the amino acid sequences, NANOBODIES and polypeptides ofthe invention can also be expressed and/or produced in cell-freeexpression systems, and suitable examples of such systems will be clearto the skilled person. Some preferred, but non-limiting examples includeexpression in the wheat germ system; in rabbit reticulocyte lysates; orin the E. coli Zubay system.

As mentioned above, one of the advantages of the use of NANOBODIES isthat the polypeptides based thereon can be prepared through expressionin a suitable bacterial system, and suitable bacterial expressionsystems, vectors, host cells, regulatory elements, etc., will be clearto the skilled person, for example from the references cited above. Itshould however be noted that the invention in its broadest sense is notlimited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expressionsystem, such as a bacterial expression system, is used that provides thepolypeptides of the invention in a form that is suitable forpharmaceutical use, and such expression systems will again be clear tothe skilled person. As also will be clear to the skilled person,polypeptides of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of NANOBODIES or NANOBODY-containing proteintherapeutics include strains of E. coli, Pichia pastoris, S. cerevisiaethat are suitable for large scale expression/production/fermentation,and in particular for large scale pharmaceutical (i.e. GMP grade)expression/production/fermentation. Suitable examples of such strainswill be clear to the skilled person. Such strains andproduction/expression systems are also made available by companies suchas Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned above.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a NANOBODY-containingrecombinant protein for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e. thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression. Preferably, either a humancell or cell line is used (i.e. leading to a protein that essentiallyhas a human glycosylation pattern) or another mammalian cell line isused that can provide a glycosylation pattern that is essentially and/orfunctionally the same as human glycosylation or at least mimics humanglycosylation. Generally, prokaryotic hosts such as E. coli do not havethe ability to glycosylate proteins, and the use of lower eukaryotessuch as yeast usually leads to a glycosylation pattern that differs fromhuman glycosylation. Nevertheless, it should be understood that all theforegoing host cells and expression systems can be used in theinvention, depending on the desired amino acid sequence, NANOBODY orpolypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, the aminoacid sequence, NANOBODY or polypeptide of the invention is glycosylated.According to another non-limiting aspect of the invention, the aminoacid sequence, NANOBODY or polypeptide of the invention isnon-glycosylated.

According to one preferred, but non-limiting aspect of the invention,the amino acid sequence, NANOBODY or polypeptide of the invention isproduced in a bacterial cell, in particular a bacterial cell suitablefor large scale pharmaceutical production, such as cells of the strainsmentioned above.

According to another preferred, but non-limiting aspect of theinvention, the amino acid sequence, NANOBODY or polypeptide of theinvention is produced in a yeast cell, in particular a yeast cellsuitable for large scale pharmaceutical production, such as cells of thespecies mentioned above.

According to yet another preferred, but non-limiting aspect of theinvention, the amino acid sequence, NANOBODY or polypeptide of theinvention is produced in a mammalian cell, in particular in a human cellor in a cell of a human cell line, and more in particular in a humancell or in a cell of a human cell line that is suitable for large scalepharmaceutical production, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the amino acidsequences, NANOBODIES and the polypeptides of the invention, the aminoacid sequences, NANOBODIES and polypeptides of the invention can beproduced either intracellullarly (e.g. in the cytosol, in the periplasmaor in inclusion bodies) and then isolated from the host cells andoptionally further purified; or can be produced extracellularly (e.g. inthe medium in which the host cells are cultured) and then isolated fromthe culture medium and optionally further purified. When eukaryotic hostcells are used, extracellular production is usually preferred since thisconsiderably facilitates the further isolation and downstream processingof the NANOBODIES and proteins obtained. Bacterial cells such as thestrains of E. coli mentioned above normally do not secrete proteinsextracellularly, except for a few classes of proteins such as toxins andhemolysin, and secretory production in E. coli refers to thetranslocation of proteins across the inner membrane to the periplasmicspace. Periplasmic production provides several advantages over cytosolicproduction. For example, the N-terminal amino acid sequence of thesecreted product can be identical to the natural gene product aftercleavage of the secretion signal sequence by a specific signalpeptidase. Also, there appears to be much less protease activity in theperiplasm than in the cytoplasm. In addition, protein purification issimpler due to fewer contaminating proteins in the periplasm. Anotheradvantage is that correct disulfide bonds may form because the periplasmprovides a more oxidative environment than the cytoplasm. Proteinsoverexpressed in E. coli are often found in insoluble aggregates,so-called inclusion bodies. These inclusion bodies may be located in thecytosol or in the periplasm; the recovery of biologically activeproteins from these inclusion bodies requires a denaturation/refoldingprocess. Many recombinant proteins, including therapeutic proteins, arerecovered from inclusion bodies. Alternatively, as will be clear to theskilled person, recombinant strains of bacteria that have beengenetically modified so as to secrete a desired protein, and inparticular an amino acid sequence, NANOBODY or a polypeptide of theinvention, can be used.

Thus, according to one non-limiting aspect of the invention, the aminoacid sequence, NANOBODY or polypeptide of the invention is an amino acidsequence, NANOBODY or polypeptide that has been produced intracellularlyand that has been isolated from the host cell, and in particular from abacterial cell or from an inclusion body in a bacterial cell. Accordingto another non-limiting aspect of the invention, the amino acidsequence, NANOBODY or polypeptide of the invention is an amino acidsequence, NANOBODY or polypeptide that has been producedextracellularly, and that has been isolated from the medium in which thehost cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cellsinclude,

-   -   for expression in E. coli: lac promoter (and derivatives thereof        such as the lacUV5 promoter); arabinose promoter; left- (PL) and        rightward (PR) promoter of phage lambda; promoter of the trp        operon; hybrid lac/trp promoters (tac and trc); T7-promoter        (more specifically that of T7-phage gene 10) and other T-phage        promoters; promoter of the Tn10 tetracycline resistance gene;        engineered variants of the above promoters that include one or        more copies of an extraneous regulatory operator sequence;    -   for expression in S. cerevisiae: constitutive: ADH1 (alcohol        dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c iso-1),        GAPDH (glyceraldehydes-3-phosphate dehydrogenase), PGK1        (phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated:        GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol        dehydrogenase 2), PHOS (acid phosphatase), CUP1 (copper        metallothionein); heterologous: CaMV (cauliflower mosaic virus        35S promoter);    -   for expression in Pichia pastoris: the AOX1 promoter (alcohol        oxidase I);    -   for expression in mammalian cells: human cytomegalovirus (hCMV)        immediate early enhancer/promoter; human cytomegalovirus (hCMV)        immediate early promoter variant that contains two tetracycline        operator sequences such that the promoter can be regulated by        the Tet repressor; Herpes Simplex Virus thymidine kinase (TK)        promoter; Rous Sarcoma Virus long terminal repeat (RSV LTR)        enhancer/promoter; elongation factor 1α (hEF-1α) promoter from        human, chimpanzee, mouse or rat; the SV40 early promoter; HIV-1        long terminal repeat promoter; β-actin promoter;

Some preferred, but non-limiting vectors for use with these host cellsinclude:

-   -   vectors for expression in mammalian cells: pMAMneo (Clontech),        pcDNA3 (Invitrogen), pMC1neo (Stratagene), pSG5 (Stratagene),        EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110),        pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo        (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and        1ZD35 (ATCC 37565), as well as viral-based expression systems,        such as those based on adenovirus;    -   vectors for expression in bacterial cells: pET vectors (Novagen)        and pQE vectors (Qiagen);    -   vectors for expression in yeast or other fungal cells: pYES2        (Invitrogen) and Pichia expression vectors (Invitrogen);    -   vectors for expression in insect cells: pBlueBacII (Invitrogen)        and other baculovirus vectors    -   vectors for expression in plants or plant cells: for example        vectors based on cauliflower mosaic virus or tobacco mosaic        virus, suitable strains of Agrobacterium, or Ti-plasmid based        vectors.

Some preferred, but non-limiting secretory sequences for use with thesehost cells include:

-   -   for use in bacterial cells such as E. coli: PelB, Bla, OmpA,        OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LamB, and the        like; TAT signal peptide, hemolysin C-terminal secretion signal;    -   for use in yeast: α-mating factor prepro-sequence, phosphatase        (phol), invertase (Suc), etc.;    -   for use in mammalian cells: indigenous signal in case the target        protein is of eukaryotic origin; murine Ig κ-chain V-J2-C signal        peptide; etc.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the amino acid sequence of the invention, e.g. usingspecific antibodies.

The transformed host cell (which may be in the form or a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), an amino acid sequence, NANOBODY or polypeptide of theinvention (and in case of a host organism: in at least one cell, part,tissue or organ thereof). The invention also includes furthergenerations, progeny and/or offspring of the host cell or host organismof the invention, that may for instance be obtained by cell division orby sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of theinvention, the transformed host cell or transformed host organism maygenerally be kept, maintained and/or cultured under conditions such thatthe (desired) amino acid sequence, NANOBODY or polypeptide of theinvention is expressed/produced. Suitable conditions will be clear tothe skilled person and will usually depend upon the host cell/hostorganism used, as well as on the regulatory elements that control theexpression of the (relevant) nucleotide sequence of the invention.Again, reference is made to the handbooks and patent applicationsmentioned above in the paragraphs on the genetic constructs of theinvention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theamino acid sequences of the invention may be expressed in a constitutivemanner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acidsequence, NANOBODY or polypeptide of the invention may (first) begenerated in an immature form (as mentioned above), which may then besubjected to post-translational modification, depending on the hostcell/host organism used. Also, the amino acid sequence, NANOBODY orpolypeptide of the invention may be glycosylated, again depending on thehost cell/host organism used.

The amino acid sequence, NANOBODY or polypeptide of the invention maythen be isolated from the host cell/host organism and/or from the mediumin which said host cell or host organism was cultivated, using proteinisolation and/or purification techniques known per se, such as(preparative) chromatography and/or electrophoresis techniques,differential precipitation techniques, affinity techniques (e.g. using aspecific, cleavable amino acid sequence fused with the amino acidsequence, NANOBODY or polypeptide of the invention) and/or preparativeimmunological techniques (i.e. using antibodies against the amino acidsequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention maybe formulated as a pharmaceutical preparation or compositions comprisingat least one polypeptide of the invention and at least onepharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activepolypeptides and/or compounds. By means of non-limiting examples, such aformulation may be in a form suitable for oral administration, forparenteral administration (such as by intravenous, intramuscular orsubcutaneous injection or intravenous infusion), for topicaladministration, for administration by inhalation, by a skin patch, by animplant, by a suppository, etc. Such suitable administration forms—whichmay be solid, semi-solid or liquid, depending on the manner ofadministration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person, and arefurther described herein.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one amino acid of the invention, atleast one NANOBODY of the invention or at least one polypeptide of theinvention and at least one suitable carrier, diluent or excipient (i.e.suitable for pharmaceutical use), and optionally one or more furtheractive substances.

Generally, the amino acid sequences, NANOBODIES and polypeptides of theinvention can be formulated and administered in any suitable mannerknown per se, for which reference is for example made to the generalbackground art cited above (and in particular to WO 04/041862, WO04/041863, WO 04/041865, WO 04/041867 and WO 08/020079) as well as tothe standard handbooks, such as Remington's Pharmaceutical Sciences,18^(th) Ed., Mack Publishing Company, USA (1990) or Remington, theScience and Practice of Pharmacy, 21st Edition, Lippincott Williams andWilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel,Ed.), Wiley, Weinheim, 2007 (see for example pages 252-255).

For example, the amino acid sequences, NANOBODIES and polypeptides ofthe invention may be formulated and administered in any manner known perse for conventional antibodies and antibody fragments (including ScFv'sand diabodies) and other pharmaceutically active proteins. Suchformulations and methods for preparing the same will be clear to theskilled person, and for example include preparations suitable forparenteral administration (for example intravenous, intraperitoneal,subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecaladministration) or for topical (i.e. transdermal or intradermal)administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, sterile water andaqueous buffers and solutions such as physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution;water oils; glycerol; ethanol; glycols such as propylene glycol or aswell as mineral oils, animal oils and vegetable oils, for example peanutoil, soybean oil, as well as suitable mixtures thereof. Usually, aqueoussolutions or suspensions will be preferred.

The amino acid sequences, NANOBODIES and polypeptides of the inventioncan also be administered using gene therapy methods of delivery. See,e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference in itsentirety. Using a gene therapy method of delivery, primary cellstransfected with the gene encoding an amino acid sequence, NANOBODY orpolypeptide of the invention can additionally be transfected with tissuespecific promoters to target specific organs, tissue, grafts, tumors, orcells and can additionally be transfected with signal and stabilizationsequences for subcellularly localized expression.

Thus, the amino acid sequences, NANOBODIES and polypeptides of theinvention may be systemically administered, e.g., orally, in combinationwith a pharmaceutically acceptable vehicle such as an inert diluent oran assimilable edible carrier. They may be enclosed in hard or softshell gelatin capsules, may be compressed into tablets, or may beincorporated directly with the food of the patient's diet. For oraltherapeutic administration, the amino acid sequences, NANOBODIES andpolypeptides of the invention may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% of theamino acid sequence, NANOBODY or polypeptide of the invention. Theirpercentage in the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the amino acidsequence, NANOBODY or polypeptide of the invention in suchtherapeutically useful compositions is such that an effective dosagelevel will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the amino acid sequences, NANOBODIES and polypeptides of theinvention, sucrose or fructose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and flavoring such as cherry ororange flavor. Of course, any material used in preparing any unit dosageform should be pharmaceutically acceptable and substantially non-toxicin the amounts employed. In addition, the amino acid sequences,NANOBODIES and polypeptides of the invention may be incorporated intosustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The amino acid sequences, NANOBODIES and polypeptides of the inventionmay also be administered intravenously or intraperitoneally by infusionor injection. Solutions of the amino acid sequences, NANOBODIES andpolypeptides of the invention or their salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the aminoacid sequences, NANOBODIES and polypeptides of the invention in therequired amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

For topical administration, the amino acid sequences, NANOBODIES andpolypeptides of the invention may be applied in pure form, i.e., whenthey are liquids. However, it will generally be desirable to administerthem to the skin as compositions or formulations, in combination with adermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, hydroxyalkyls or glycols or water-alcohol/glycolblends, in which the amino acid sequences, NANOBODIES and polypeptidesof the invention can be dissolved or dispersed at effective levels,optionally with the aid of non-toxic surfactants. Adjuvants such asfragrances and additional antimicrobial agents can be added to optimizethe properties for a given use. The resultant liquid compositions can beapplied from absorbent pads, used to impregnate bandages and otherdressings, or sprayed onto the affected area using pump-type or aerosolsprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the amino acid sequences, NANOBODIES and polypeptides of theinvention to the skin are known to the art; for example, see Jacquet etal. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith etal. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the amino acid sequences, NANOBODIES and polypeptidesof the invention can be determined by comparing their in vitro activity,and in vivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the amino acid sequences, NANOBODIES andpolypeptides of the invention in a liquid composition, such as a lotion,will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. Theconcentration in a semi-solid or solid composition such as a gel or apowder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the amino acid sequences, NANOBODIES and polypeptides ofthe invention required for use in treatment will vary not only with theparticular amino acid sequence, NANOBODY or polypeptide selected butalso with the route of administration, the nature of the condition beingtreated and the age and condition of the patient and will be ultimatelyat the discretion of the attendant physician or clinician. Also thedosage of the amino acid sequences, NANOBODIES and polypeptides of theinvention varies depending on the target cell, tumor, tissue, graft, ororgan.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one bone disease or disorder, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a NANOBODY of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention relates to a method for the prevention and/or treatment ofat least one disease or disorder that is associated with RANK-L, withits biological or pharmacological activity, and/or with the biologicalpathways or signalling in which RANK-L is involved, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a NANOBODY of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.In particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder that can be treatedby modulating RANK-L, its biological or pharmacological activity, and/orthe biological pathways or signalling in which RANK-L is involved, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a NANOBODY of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.In particular, said pharmaceutically effective amount may be an amountthat is sufficient to modulate RANK-L, its biological or pharmacologicalactivity, and/or the biological pathways or signalling in which RANK-Lis involved; and/or an amount that provides a level of the amino acidsequence of the invention, of a NANOBODY of the invention, of apolypeptide of the invention in the circulation that is sufficient tomodulate RANK-L, its biological or pharmacological activity, and/or thebiological pathways or signalling in which RANK-L is involved.

The invention furthermore relates to a method for the prevention and/ortreatment of at least one disease or disorder that can be preventedand/or treated by administering an amino acid sequence of the invention,a NANOBODY of the invention or a polypeptide of the invention to apatient, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of an amino acid sequence ofthe invention, of a NANOBODY of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a NANOBODY of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In another aspect, the invention relates to a method for immunotherapy,and in particular for passive immunotherapy, which method comprisesadministering, to a subject suffering from or at risk of the diseasesand disorders mentioned herein, a pharmaceutically active amount of anamino acid sequence of the invention, of a NANOBODY of the invention, ofa polypeptide of the invention, and/or of a pharmaceutical compositioncomprising the same.

In the above methods, the amino acid sequences, NANOBODIES and/orpolypeptides of the invention and/or the compositions comprising thesame can be administered in any suitable manner, depending on thespecific pharmaceutical formulation or composition to be used. Thus, theamino acid sequences, NANOBODIES and/or polypeptides of the inventionand/or the compositions comprising the same can for example beadministered orally, intraperitoneally (e.g. intravenously,subcutaneously, intramuscularly, or via any other route ofadministration that circumvents the gastrointestinal tract),intranasally, transdermally, topically, by means of a suppository, byinhalation, again depending on the specific pharmaceutical formulationor composition to be used. The clinician will be able to select asuitable route of administration and a suitable pharmaceuticalformulation or composition to be used in such administration, dependingon the disease or disorder to be prevented or treated and other factorswell known to the clinician.

The amino acid sequences, NANOBODIES and/or polypeptides of theinvention and/or the compositions comprising the same are administeredaccording to a regime of treatment that is suitable for preventingand/or treating the disease or disorder to be prevented or treated. Theclinician will generally be able to determine a suitable treatmentregimen, depending on factors such as the disease or disorder to beprevented or treated, the severity of the disease to be treated and/orthe severity of the symptoms thereof, the specific amino acid sequence,NANOBODY or polypeptide of the invention to be used, the specific routeof administration and pharmaceutical formulation or composition to beused, the age, gender, weight, diet, general condition of the patient,and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more amino acid sequences, NANOBODIES and/or polypeptides of theinvention, or of one or more compositions comprising the same, in one ormore pharmaceutically effective amounts or doses. The specific amount(s)or doses to administer can be determined by the clinician, again basedon the factors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific amino acid sequence,NANOBODY and polypeptide of the invention to be used, the specific routeof administration and the specific pharmaceutical formulation orcomposition used, the amino acid sequences, NANOBODIES and polypeptidesof the invention will generally be administered in an amount between 1gram and 0.01 microgram per kg body weight per day, preferably between0.1 gram and 0.1 microgram per kg body weight per day, such as about 1,10, 100 or 1000 microgram per kg body weight per day, eithercontinuously (e.g. by infusion), as a single daily dose or as multipledivided doses during the day. The clinician will generally be able todetermine a suitable daily dose, depending on the factors mentionedherein. It will also be clear that in specific cases, the clinician maychoose to deviate from these amounts, for example on the basis of thefactors cited above and his expert judgment. Generally, some guidance onthe amounts to be administered can be obtained from the amounts usuallyadministered for comparable conventional antibodies or antibodyfragments against the same target administered via essentially the sameroute, taking into account however differences in affinity/avidity,efficacy, biodistribution, half-life and similar factors well known tothe skilled person.

Usually, in the above method, a single amino acid sequence, NANOBODY orpolypeptide of the invention will be used. It is however within thescope of the invention to use two or more amino acid sequences,NANOBODIES and/or polypeptides of the invention in combination.

The NANOBODIES, amino acid sequences and polypeptides of the inventionmay also be used in combination with one or more furtherpharmaceutically active compounds or principles, i.e. as a combinedtreatment regimen, which may or may not lead to a synergistic effect.Again, the clinician will be able to select such further compounds orprinciples, as well as a suitable combined treatment regimen, based onthe factors cited above and his expert judgement.

In particular, the amino acid sequences, NANOBODIES and polypeptides ofthe invention may be used in combination with other pharmaceuticallyactive compounds or principles that are or can be used for theprevention and/or treatment of the diseases and disorders cited herein,as a result of which a synergistic effect may or may not be obtained.Examples of such compounds and principles, as well as routes, methodsand pharmaceutical formulations or compositions for administering themwill be clear to the clinician.

In particular, the pharmaceutical composition of the invention maycomprise one or more amino acid sequences, NANOBODIES and/orpolypeptides of the invention and at least one additional therapeuticagent selected from a bone morphogenic factor, transforming growthfactor-β (TGF-β), an interleukin-1 (IL-1) inhibitor, IL-1ra, Kineret™, aTNFα inhibitor, a soluble TNFα receptor, Enbrel™, an anti-TNFα antibody,Remicade™, a D2E7 antibody, a parathyroid hormone, an analog of aparathyroid hormone, a parathyroid hormone related protein, an analog ofa parathyroid hormone related protein, a prostaglandin, abisphosphonate, an alendronate, fluoride, calcium, a non-steroidalanti-inflammatory drug (NSAID), a COX-2 inhibitor, Celebrex™, Vioxx™, animmunosuppressant, methotrexate, leflunomide, a serine proteaseinhibitor, a secretory leukocyte protease inhibitor (SLPI), an IL-6inhibitor, an antibody or NANOBODY against IL-6, an IL-8 inhibitor, anantibody or NANOBODY against IL-8, an IL-18 inhibitor, an IL-18 bindingprotein, an antibody or NANOBODY against IL-18, an Interleukin-1converting enzyme (ICE) modulator, a fibroblast growth factor (FGF), anFGF modulator, a PAF antagonist, a keratinocyte growth factor (KGF), aKGF-related molecule, a KGF modulator, a matrix metalloproteinase (MMP)modulator, a nitric oxide synthase (NOS) modulator, a modulator ofglucocorticoid receptor, a modulator of glutamate receptor, a modulatorof lipopolysaccharide (LPS) levels, a noradrenaline, a noradrenalinemimetic, and a noradrenaline modulator as described, for example, in US2004/00335353

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and on a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

In another aspect, the invention relates to the use of an amino acidsequence, NANOBODY or polypeptide of the invention in the preparation ofa pharmaceutical composition for prevention and/or treatment of at leastone bone disease or disorder; and/or for use in one or more of themethods of treatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention also relates to the use of an amino acid sequence,NANOBODY or polypeptide of the invention in the preparation of apharmaceutical composition for the prevention and/or treatment of atleast one disease or disorder that can be prevented and/or treated byadministering an amino acid sequence, NANOBODY or polypeptide of theinvention to a patient.

More in particular, the invention relates to the use of an amino acidsequence, NANOBODY or polypeptide of the invention in the preparation ofa pharmaceutical composition for the prevention and/or treatment of bonediseases and disorders, and in particular for the prevention andtreatment of one or more of the diseases and disorders listed herein.

Again, in such a pharmaceutical composition, the one or more amino acidsequences, NANOBODIES or polypeptides of the invention may also besuitably combined with one or more other active principles, such asthose mentioned herein.

Finally, although the use of the NANOBODIES of the invention (as definedherein) and of the polypeptides of the invention is much preferred, itwill be clear that on the basis of the description herein, the skilledperson will also be able to design and/or generate, in an analogousmanner, other amino acid sequences and in particular (single) domainantibodies against RANK-L, as well as polypeptides comprising such(single) domain antibodies.

For example, it will also be clear to the skilled person that it may bepossible to “graft” one or more of the CDR's mentioned above for theNANOBODIES of the invention onto such (single) domain antibodies orother protein scaffolds, including but not limited to human scaffolds ornon-immunoglobulin scaffolds. Suitable scaffolds and techniques for suchCDR grafting will be clear to the skilled person and are well known inthe art, see for example U.S. Pat. No. 7,180,370, WO 01/27160, EP 0 605522, EP 0 460 167, U.S. Pat. No. 7,054,297, Nicaise et al., ProteinScience (2004), 13:1882-1891; Ewert et al., Methods, 2004 October;34(2):184-199; Kettleborough et al., Protein Eng. 1991 October; 4(7):773-783; O'Brien and Jones, Methods Mol. Biol. 2003: 207: 81-100;Skerra, J. Mol. Recognit. 2000: 13: 167-187, and Saerens et al., J. Mol.Biol. 2005 Sep. 23; 352(3):597-607, and the further references citedtherein. For example, techniques known per se for grafting mouse or ratCDR's onto human frameworks and scaffolds can be used in an analogousmanner to provide chimeric proteins comprising one or more of the CDR'sof the NANOBODIES of the invention and one or more human frameworkregions or sequences.

It should also be noted that, when the NANOBODIES of the inventionscontain one or more other CDR sequences than the preferred CDR sequencesmentioned above, these CDR sequences can be obtained in any manner knownper se, for example from NANOBODIES (preferred), V_(H) domains fromconventional antibodies (and in particular from human antibodies), heavychain antibodies, conventional 4-chain antibodies (such as conventionalhuman 4-chain antibodies) or other immunoglobulin sequences directedagainst RANK-L. Such immunoglobulin sequences directed against RANK-Lcan be generated in any manner known per se, as will be clear to theskilled person, i.e. by immunization with RANK-L or by screening asuitable library of immunoglobulin sequences with RANK-L, or anysuitable combination thereof. Optionally, this may be followed bytechniques such as random or site-directed mutagenesis and/or othertechniques for affinity maturation known per se. Suitable techniques forgenerating such immunoglobulin sequences will be clear to the skilledperson, and for example include the screening techniques reviewed byHoogenboom, Nature Biotechnology, 23, 9, 1105-1116 (2005). Othertechniques for generating immunoglobulins against a specified targetinclude for example the Nanoclone technology (as for example describedin the published US patent application 2006-0211088), so-called SLAMtechnology (as for example described in the European patent application0 542 810), the use of transgenic mice expressing human immunoglobulinsor the well-known hybridoma techniques (see for example Larrick et al,Biotechnology, Vol. 7, 1989, p. 934). All these techniques can be usedto generate immunoglobulins against RANK-L, and the CDR's of suchimmunoglobulins can be used in the NANOBODIES of the invention, i.e. asoutlined above. For example, the sequence of such a CDR can bedetermined, synthesized and/or isolated, and inserted into the sequenceof a NANOBODY of the invention (e.g. so as to replace the correspondingnative CDR), all using techniques known per se such as those describedherein, or NANOBODIES of the invention containing such CDR's (or nucleicacids encoding the same) can be synthesized de novo, again using thetechniques mentioned herein.

Further uses of the amino acid sequences, NANOBODIES, polypeptides,nucleic acids, genetic constructs and hosts and host cells of theinvention will be clear to the skilled person based on the disclosureherein. For example, and without limitation, the amino acid sequences ofthe invention can be linked to a suitable carrier or solid support so asto provide a medium than can be used in a manner known per se to purifyRANK-L from compositions and preparations comprising the same.Derivatives of the amino acid sequences of the invention that comprise asuitable detectable label can also be used as markers to determine(qualitatively or quantitatively) the presence of RANK-L in acomposition or preparation or as a marker to selectively detect thepresence of RANK-L on the surface of a cell or tissue (for example, incombination with suitable cell sorting techniques).

The invention will now be further described by means of the followingnon-limiting preferred aspects, examples and figures:

Preferred Aspects

-   1. Amino acid sequence that is directed against and/or that can    specifically bind to RANK-L.-   2. Amino acid sequence according to aspect 1, which is directed    against and/or can specifically bind to the RANK receptor binding    site on RANK-L.-   3. Amino acid sequence according to any of aspects 1 or 2, which is    directed against and/or can specifically bind to the intersubunit    receptor-binding grooves on the RANK-L trimer.-   4. Amino acid sequence according to any of aspects 1 to 3, which    modulates binding of RANKL-L to RANK.-   5. Amino acid sequence according to aspect 4, which inhibits and/or    prevents binding of RANKL-L to RANK.-   6. Amino acid sequence according to aspect 5, which inhibits and/or    prevents binding of RANKL-L to RANK, while not reducing and/or    inhibiting the RANK-L/OPG interaction.-   7. Amino acid sequence according to any of aspects 1 to 6, which is    an antagonist of RANK-L-   8. Amino acid sequence according aspect 1, which is directed against    and/or can specifically bind to the OPG binding site on RANK-L.-   9. Amino acid sequence according aspects 1 or 8, which modulates    binding of RANKL-L to OPG.-   10 Amino acid sequence according to aspect 9, which inhibits and/or    prevents the RANK/RANK-L interaction.-   11. Amino acid sequence according to aspect 10, which is an    antagonist of RANK-L.-   12 Amino acid sequence according to aspect 8, which does not reduce    or inhibit the RANK/RANK-L interaction.-   13. Amino acid sequence according to aspect 12, which is an agonist    of RANK-L.-   14 Amino acid sequence according to aspect 1, which prevents and/or    inhibits the formation of the RANK-L trimer.-   15 Amino acid sequence according to aspect 1, which prevents and/or    inhibits the differentiation and/or proliferation of osteoclasts.-   16. Amino acid sequence according to aspect 1, which modulates bone    remodelling.-   17. Amino acid sequence according to any of aspects 1 to 16, which    does not bind TRAIL.-   18 Amino acid sequence according to any of aspects 1 to 17, which    does not bind TNF-alpha.-   19 Amino acid sequence according to any of aspects 1 to 18, which    does not bind CD40 ligand.-   20 Amino acid sequence according to any of aspects 1 to 19, which    does not bind related TNF family members.-   21 Amino acid sequence according to any of aspects 1 to 20, that is    in essentially isolated form.-   22 Amino acid sequence according to any of aspects 1 to 21, for    administration to a subject, wherein said amino acid sequence does    not naturally occur in said subject.-   23 Amino acid sequence according to any of the preceding aspects,    that can specifically bind to RANK-L with a dissociation constant    (K_(D)) of 10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to    10⁻¹² moles/litre or less and more preferably 10⁻⁸ to 10⁻¹²    moles/litre.-   24 Amino acid sequence according to any of the preceding aspects,    that can specifically bind to RANK-L with a rate of association    (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably    between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.-   25 Amino acid sequence according to any of the preceding aspects,    that can specifically bind to RANK-L with a rate of dissociation    (k_(off) rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹, preferably between 10⁻²    s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹,    such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   26 Amino acid sequence according to any of the preceding aspects,    that can specifically bind to RANK-L with an affinity less than 500    nM, preferably less than 200 nM, more preferably less than 10 nM,    such as less than 500 pM.-   27 Amino acid sequence according to any of the preceding aspects,    that is a naturally occurring amino acid sequence (from any suitable    species) or a synthetic or semi-synthetic amino acid sequence.-   28 Amino acid sequence according to any of the preceding aspects,    that comprises an immunoglobulin fold or that under suitable    conditions is capable of forming an immunoglobulin fold.-   29 Amino acid sequence according to any of the preceding aspects,    that essentially consists of 4 framework regions (FR1 to FR4    respectively) and 3 complementarity determining regions (CDR1 to    CDR3 respectively).-   30 Amino acid sequence according to any of the preceding aspects,    that is an immunoglobulin sequence.-   31 Amino acid sequence according to any of the preceding aspects,    that is a naturally occurring immunoglobulin sequence (from any    suitable species) or a synthetic or semi-synthetic immunoglobulin    sequence.-   32 Amino acid sequence according to any of the preceding aspects    that is a humanized immunoglobulin sequence, a camelized    immunoglobulin sequence or an immunoglobulin sequence that has been    obtained by techniques such as affinity maturation.-   33 Amino acid sequence according to any of the preceding aspects,    that essentially consists of a light chain variable domain sequence    (e.g. a V_(L)-sequence); or of a heavy chain variable domain    sequence (e.g. a V_(H)-sequence).-   34 Amino acid sequence according to any of the preceding aspects,    that essentially consists of a heavy chain variable domain sequence    that is derived from a conventional four-chain antibody or that    essentially consist of a heavy chain variable domain sequence that    is derived from heavy chain antibody.-   35 Amino acid sequence according to any of the preceding aspects,    that essentially consists of a domain antibody (or an amino acid    sequence that is suitable for use as a domain antibody), of a single    domain antibody (or an amino acid sequence that is suitable for use    as a single domain antibody), of a “dAb” (or an amino acid sequence    that is suitable for use as a dAb) or of a NANOBODY (including but    not limited to a V_(HH) sequence).-   36 Amino acid sequence according to any of the preceding aspects,    that essentially consists of a NANOBODY.-   37 Amino acid sequence according to any of the preceding aspects,    that essentially consists of a NANOBODY that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 1 to 22, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;

and in which:

-   -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.

-   38 Amino acid sequence according to any of the preceding aspects,    that essentially consists of a NANOBODY that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 560-621, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;

and in which:

-   -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.

-   39 Amino acid sequence according to any of the preceding aspects,    that essentially consists of a polypeptide that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 622-729, 759-762 and        766-773, in which for the purposes of determining the degree of        amino acid identity, the amino acid residues that form the CDR        sequences are disregarded;

and in which:

-   -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.

-   40 Amino acid sequence according to any of the preceding aspects,    that essentially consists of a humanized NANOBODY.

-   41 Amino acid sequence according to any of the preceding aspects,    that in addition to the at least one binding site for binding    against RANK-L, contains one or more further binding sites for    binding against other antigens, proteins or targets.

-   42 Amino acid sequence directed against RANK-L, that comprises one    or more stretches of amino acid residues chosen from the group    consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 188-249;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 188-249;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 188-249;    -   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 312-373 and 758;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 312-373 and 758;    -   g) the amino acid sequences of SEQ ID NO's: 436-497;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 436-497;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 436-497;    -   or any suitable combination thereof.

-   43 Amino acid sequence according to aspect 42, in which at least one    of said stretches of amino acid residues forms part of the antigen    binding site for binding against RANK-L.

-   44 Amino acid sequence according to aspect 42, that comprises two or    more stretches of amino acid residues chosen from the group    consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 188-249;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 188-249;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 188-249;    -   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 312-373 and 758;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 312-373 and 758;    -   g) the amino acid sequences of SEQ ID NO's: 436-497;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 436-497;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 439-497;    -   such that (i) when the first stretch of amino acid residues        corresponds to one of the amino acid sequences according to        a), b) or c), the second stretch of amino acid residues        corresponds to one of the amino acid sequences according to d),        e), f), g), h) or i); (ii) when the first stretch of amino acid        residues corresponds to one of the amino acid sequences        according to d), e) or f), the second stretch of amino acid        residues corresponds to one of the amino acid sequences        according to a), b), c), g), h) or i); or (iii) when the first        stretch of amino acid residues corresponds to one of the amino        acid sequences according to g), h) or i), the second stretch of        amino acid residues corresponds to one of the amino acid        sequences according to a), b), c), d), e) or f).

-   45 Amino acid sequence according to aspect 44, in which the at least    two stretches of amino acid residues forms part of the antigen    binding site for binding against RANK-L.

-   46 Amino acid sequence according to any of aspects 42 to 44, that    comprises three or more stretches of amino acid residues, in which    the first stretch of amino acid residues is chosen from the group    consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 188-249;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 188-249;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 188-249;    -   the second stretch of amino acid residues is chosen from the        group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 312-373 and 758;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 312-373 and 758;    -   and the third stretch of amino acid residues is chosen from the        group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 436-497;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 436-497;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 436-497.

-   47 Amino acid sequence according to aspect 46, in which the at least    three stretches of amino acid residues forms part of the antigen    binding site for binding against RANK-L.

-   48. Amino acid sequence according to any of aspects 42 to 47, in    which the CDR sequences of said amino acid sequence have at least    70% amino acid identity, preferably at least 80% amino acid    identity, more preferably at least 90% amino acid identity, such as    95% amino acid identity or more or even essentially 100% amino acid    identity with the CDR sequences of at least one of the amino acid    sequences of SEQ ID NO's: 560-621.

-   49 Amino acid sequence directed against RANK-L that cross-blocks the    binding of at least one of the amino acid sequences according to any    of aspects 42 to 48 to RANK-L.

-   50 Amino acid sequence directed against RANK-L that is cross-blocked    from binding to RANK-L by at least one of the amino acid sequences    according to any of aspects 42 to 48.

-   51 Amino acid sequence according to any of aspects 49 or 50 wherein    the ability of said amino acid sequence to cross-block or to be    cross-blocked is detected in a BIACORE assay.

-   52 Amino acid sequence according to any of aspects 49 or 50 wherein    the ability of said amino acid sequence to cross-block or to be    cross-blocked is detected in an ELISA assay.

-   53 Amino acid sequence according to any of aspects 42 to 52, that is    in essentially isolated form.

-   54 Amino acid sequence according to any of aspects 42 to 53, for    administration to a subject, wherein said amino acid sequence does    not naturally occur in said subject.

-   55 Amino acid sequence according to any of aspects 42 to 54, that    can specifically bind to RANK-L with a dissociation constant (K_(D))    of 10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²    moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre.

-   56 Amino acid sequence according to any of aspects 42 to 55, that    can specifically bind to RANK-L with a rate of association    (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably    between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.

-   57 Amino acid sequence according to any of aspects 42 to 56, that    can specifically bind to RANK-L with a rate of dissociation (k_(off)    rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and    10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as    between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

-   58 Amino acid sequence according to any of aspects 42 to 57, that    can specifically bind to RANK-L with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM.

-   59 Amino acid sequence according to any of aspects 42 to 58, that is    a naturally occurring amino acid sequence (from any suitable    species) or a synthetic or semi-synthetic amino acid sequence.

-   60 Amino acid sequence according to any of aspects 42 to 59, that    comprises an immunoglobulin fold or that under suitable conditions    is capable of forming an immunoglobulin fold.

-   61 Amino acid sequence according to any of aspects 42 to 60, that is    an immunoglobulin sequence.

-   62 Amino acid sequence according to any of aspects 42 to 61 that is    a naturally occurring immunoglobulin sequence (from any suitable    species) or a synthetic or semi-synthetic immunoglobulin sequence.

-   63 Amino acid sequence according to any of aspects 42 to 62, that is    a humanized immunoglobulin sequence, a camelized immunoglobulin    sequence or an immunoglobulin sequence that has been obtained by    techniques such as affinity maturation.

-   64. Amino acid sequence according to any of aspects 42 to 63, that    essentially consists of a light chain variable domain sequence (e.g.    a V_(L)-sequence); or of a heavy chain variable domain sequence    (e.g. a V_(H)-sequence).

-   65 Amino acid sequence according to any of aspects 42 to 64, that    essentially consists of a heavy chain variable domain sequence that    is derived from a conventional four-chain antibody or that    essentially consist of a heavy chain variable domain sequence that    is derived from heavy chain antibody.

-   66 Amino acid sequence according to any of aspects 42 to 65, that    essentially consists of a domain antibody (or an amino acid sequence    that is suitable for use as a domain antibody), of a single domain    antibody (or an amino acid sequence that is suitable for use as a    single domain antibody), of a “dAb” (or an amino acid sequence that    is suitable for use as a dAb) or of a NANOBODY (including but not    limited to a V_(HH) sequence).

-   67 Amino acid sequence according to any of aspects 42 to 66, that    essentially consists of a NANOBODY.

-   68 Amino acid sequence according to any of aspects 42 to 67, that    essentially consists of a NANOBODY that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 1 to 22, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;

and in which:

-   -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.

-   69 Amino acid sequence according to any of aspects 41 to 63, that    essentially consists of a NANOBODY that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 560-621, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;

and in which:

-   -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.

-   70 Amino acid sequence according to any of aspects 42 to 69, that    essentially consists of a humanized NANOBODY.

-   71 Amino acid sequence according to any of the preceding aspects,    that in addition to the at least one binding site for binding formed    by the CDR sequences, contains one or more further binding sites for    binding against other antigens, proteins or targets.

-   72 Amino acid sequence that essentially consists of 4 framework    regions (FR1 to FR4, respectively) and 3 complementarity determining    regions (CDR1 to CDR3, respectively), in which:    -   CDR1 is chosen from the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 188-249;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 188-249;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 188-249;    -   and/or    -   CDR2 is chosen from the group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 312-373 and 758;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 312-373 and 758;    -   and/or    -   CDR3 is chosen from the group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 436-497;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 436-497;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 436-497.

-   73 Amino acid sequence that essentially consists of 4 framework    regions (FR1 to FR4, respectively) and 3 complementarity determining    regions (CDR1 to CDR3, respectively), in which:    -   CDR1 is chosen from the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 188-249;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 188-249;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 188-249;    -   and    -   CDR2 is chosen from the group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 312-373 and 758;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 312-373 and 758;    -   and    -   CDR3 is chosen from the group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 436-497;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 436-497;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 436-497.

-   74 Amino acid sequence according to any of aspects 72 to 73, in    which the CDR sequences of said amino acid sequence have at least    70% amino acid identity, preferably at least 80% amino acid    identity, more preferably at least 90% amino acid identity, such as    95% amino acid identity or more or even essentially 100% amino acid    identity with the CDR sequences of at least one of the amino acid    sequences of SEQ ID NO's: 560-621.

-   75 Amino acid sequence directed against RANK-L that cross-blocks the    binding of at least one of the amino acid sequences according to any    of aspects 72 to 74 to RANK-L.

-   76 Amino acid sequence directed against RANK-L that is cross-blocked    from binding to RANK-L by at least one of the amino acid sequences    according to any of aspects 72 to 74.

-   77 Amino acid sequence according to any of aspects 75 or 76 wherein    the ability of said amino acid sequence to cross-block or to be    cross-blocked is detected in a BIACORE assay.

-   78 Amino acid sequence according to any of aspects 75 or 76 wherein    the ability of said amino acid sequence to cross-block or to be    cross-blocked is detected in an ELISA assay.

-   79 Amino acid sequence according to any of aspects 72 to 78, that is    in essentially isolated form.

-   80 Amino acid sequence according to any of aspects 72 to 79, for    administration to a subject, wherein said amino acid sequence does    not naturally occur in said subject.

-   81 Amino acid sequence according to any of aspects 72 to 80, that    can specifically bind to RANK-L with a dissociation constant (K_(D))    of 10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²    moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre.

-   82 Amino acid sequence according to any of aspects 72 to 81, that    can specifically bind to RANK-L with a rate of association    (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably    between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.

-   83 Amino acid sequence according to any of aspects 72 to 82, that    can specifically bind to RANK-L with a rate of dissociation (k_(off)    rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and    10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as    between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

-   84 Amino acid sequence according to any of aspects 72 to 83, that    can specifically bind to RANK-L with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM.

-   85 Amino acid sequence according to any of aspects 72 to 84, that is    a naturally occurring amino acid sequence (from any suitable    species) or a synthetic or semi-synthetic amino acid sequence.

-   86 Amino acid sequence according to any of aspects 72 to 85, that    comprises an immunoglobulin fold or that under suitable conditions    is capable of forming an immunoglobulin fold.

-   87 Amino acid sequence according to any of aspects 72 to 86, that is    an immunoglobulin sequence.

-   88 Amino acid sequence according to any of aspects 72 to 87, that is    a naturally occurring immunoglobulin sequence (from any suitable    species) or a synthetic or semi-synthetic immunoglobulin sequence.

-   89 Amino acid sequence according to any of aspects 72 to 88, that is    a humanized immunoglobulin sequence, a camelized immunoglobulin    sequence or an immunoglobulin sequence that has been obtained by    techniques such as affinity maturation.

-   90. Amino acid sequence according to any of aspects 73 to 89, that    essentially consists of a light chain variable domain sequence (e.g.    a V_(L)-sequence); or of a heavy chain variable domain sequence    (e.g. a V_(H)-sequence).

-   91 Amino acid sequence according to any of aspects 72 to 90, that    essentially consists of a heavy chain variable domain sequence that    is derived from a conventional four-chain antibody or that    essentially consist of a heavy chain variable domain sequence that    is derived from heavy chain antibody.

-   92 Amino acid sequence according to any of aspects 72 to 91, that    essentially consists of a domain antibody (or an amino acid sequence    that is suitable for use as a domain antibody), of a single domain    antibody (or an amino acid sequence that is suitable for use as a    single domain antibody), of a “dAb” (or an amino acid sequence that    is suitable for use as a dAb) or of a NANOBODY (including but not    limited to a V_(HH) sequence).

-   93 Amino acid sequence according to any of aspects 72 to 92, that    essentially consists of a NANOBODY.

-   94 Amino acid sequence according to any of aspects 72 to 93, that    essentially consists of a NANOBODY that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 1 to 22, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;

and in which:

-   -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.

-   95 Amino acid sequence according to any of aspects 72 to 94, that    essentially consists of a NANOBODY that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 560-621, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;

and in which:

-   -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.

-   96 Amino acid sequence according to any of aspects 72 to 95, that    essentially consists of a humanized NANOBODY.

-   97 Amino acid sequence according to any of the preceding aspects,    that in addition to the at least one binding site for binding formed    by the CDR sequences, contains one or more further binding sites for    binding against other antigens, proteins or targets.

-   98. NANOBODY that is directed against and/or that can specifically    bind to RANK-L.

-   99. NANOBODY according to aspect 98, which is directed against    and/or can specifically bind to the RANK receptor binding site on    RANK-L.

-   100. NANOBODY according to any of aspects 98 or 99, which is    directed against and/or can specifically bind to the intersubunit    receptor-binding grooves on the RANK-L trimer.

-   101. NANOBODY according to any of aspects 98 to 100, which modulates    binding of RANKL-L to RANK.

-   102. NANOBODY according to aspect 101, which inhibits and/or    prevents binding of RANKL-L to RANK.

-   103. NANOBODY according to aspect 102, which inhibits and/or    prevents binding of RANKL-L to RANK, while not reducing and/or    inhibiting the RANK-L/OPG interaction.

-   104. NANOBODY according to any of aspects 98 to 103, which is an    antagonist of RANK-L.

-   105. NANOBODY according aspect 98, which is directed against and/or    can specifically bind to the OPG binding site on RANK-L.

-   106. NANOBODY according aspects 98 or 105, which modulates binding    of RANKL-L to OPG.

-   107. NANOBODY according to aspect 106, which inhibits the    RANK/RANK-L interaction.

-   108. NANOBODY according to aspect 107, which is an antagonist of    RANK-L.

-   109. NANOBODY according to aspect 106, which does not reduce or    inhibit the RANK/RANK-L interaction.

-   110. NANOBODY according to aspect 109, which is an agonist of    RANK-L.

-   111. NANOBODY according to aspect 98, which prevents and/or inhibits    the formation of the RANK-L trimer.

-   112. NANOBODY according to aspect 98, which prevents and/or inhibits    the differentiation and/or proliferation of osteoclasts.

-   113. NANOBODY according to aspect 98, which modulates bone    remodelling.

-   114. NANOBODY according to any of aspects 98 to 113, which does not    bind TRAIL.

-   115. NANOBODY according to any of aspects 98 to 114, which does not    bind TNF-alpha.

-   116. NANOBODY according to any of aspects 98 to 115, which does not    bind CD40 ligand.

-   117. NANOBODY according to any of aspects 98 to 116, which does not    bind related TNF family members.

-   118. NANOBODY according to any of aspects 98 to 117, that is in    essentially isolated form.

-   119. NANOBODY according to any of aspects 98 to 118, that can    specifically bind to RANK-L with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²    moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre.

-   120. NANOBODY according to any of aspects 98 to 119, that can    specifically bind to RANK-L with a rate of association (k_(on)-rate)    of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷    M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.

-   121. NANOBODY according to any of aspects 98 to 120, that can    specifically bind to RANK-L with a rate of dissociation (k_(off)    rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and    10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as    between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

-   122. NANOBODY according to any of aspects 98 to 121, that can    specifically bind to RANK-L with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM.

-   123. NANOBODY according to any of aspects 98 to 122, that is a    naturally occurring NANOBODY (from any suitable species) or a    synthetic or semi-synthetic NANOBODY.

-   124. NANOBODY according to any of aspects 98 to 123 that is a V_(HH)    sequence, a partially humanized V_(HH) sequence, a fully humanized    V_(HH) sequence, a camelized heavy chain variable domain or a    NANOBODY that has been obtained by techniques such as affinity    maturation.

-   125. NANOBODY according to any of aspects 98 to 124, that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 1 to 22, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.

-   126. NANOBODY according to any of aspects 98 to 125, that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 560-621, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.

-   127. NANOBODY according to any of aspects 98 to 126, in which:    -   CDR1 is chosen from the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 188-249;

b) amino acid sequences that have at least 80% amino acid identity withat least one of the amino acid sequences of SEQ ID NO's: 188-249;

c) amino acid sequences that have 3, 2, or 1 amino acid difference withat least one of the amino acid sequences of SEQ ID NO's: 188-249;

-   -   and/or    -   CDR2 is chosen from the group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 312-373 and 758;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 312-373 and 758;    -   and/or    -   CDR3 is chosen from the group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 436-497;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 436-497;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 436-497.

-   128. NANOBODY according to any of aspects 98 to 129, in which:    -   CDR1 is chosen from the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 188-249;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 188-249;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 188-249;    -   and    -   CDR2 is chosen from the group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 312-373 and 758;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 312-373 and 758;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 312-373 and 758;    -   and    -   CDR3 is chosen from the group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 436-497;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 436-497;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 439-497.

-   129. NANOBODY according to any of aspects 98 to 128, in which the    CDR sequences have at least 70% amino acid identity, preferably at    least 80% amino acid identity, more preferably at least 90% amino    acid identity, such as 95% amino acid identity or more or even    essentially 100% amino acid identity with the CDR sequences of at    least one of the amino acid sequences of SEQ ID NO's: 560-621.

-   130. NANOBODY according to any of aspects 98 to 129, which is a    partially humanized NANOBODY.

-   131. NANOBODY according to any of aspects 98 to 130, which is a    fully humanized NANOBODY.

-   132. NANOBODY according to any of aspects 98 to 131, that is chosen    from the group consisting of SEQ ID NO's: 560-621 or from the group    consisting of from amino acid sequences that have more than 80%,    preferably more than 90%, more preferably more than 95%, such as 99%    or more sequence identity (as defined herein) with at least one of    the amino acid sequences of SEQ ID NO's: 560-621.

-   133. NANOBODY according to any of aspects 98 to 131, which is a    humanized NANOBODY that is chosen from the group consisting of SEQ    ID NO's: 730-757 and 765 or from the group consisting of from amino    acid sequences that have more than 80%, preferably more than 90%,    more preferably more than 95%, such as 99% or more sequence identity    (as defined herein) with at least one of the amino acid sequences of    SEQ ID NO's: 730-757 and 765.

-   134. NANOBODY according to any of aspects 98 to 133, that is chosen    from the group consisting of SEQ ID NO's: 560-621 or from the group    consisting of SEQ ID NO's: 730-757 and 765.

-   135. NANOBODY directed against RANK-L that cross-blocks the binding    of at least one of the amino acid sequences according to any of    aspects 42 to 48 or 72 to 74 or NANOBODIES according to any of    aspects 127 to 134 to RANK-L.

-   136. NANOBODY directed against RANK-L that is cross-blocked from    binding to RANK-L by at least one of the amino acid sequences    according to any of aspects 42 to 48 or 72 to 74 or NANOBODIES    according to any of aspects 127 to 143.

-   137. NANOBODY according to any of aspects 135 or 136 wherein the    ability of said NANOBODY to cross-block or to be cross-blocked is    detected in a BIACORE assay.

-   138. NANOBODY according to any of aspects 135 or 136 wherein the    ability of said NANOBODY to cross-block or to be cross-blocked is    detected in an ELISA assay.

-   139. Polypeptide that comprises or essentially consists of one or    more amino acid sequences according to any of aspects 1 to 97 and/or    one or more NANOBODIES according to any of aspects 98 to 138, and    optionally further comprises one or more other amino acid binding    units, optionally linked via one or more peptidic linkers.

-   140. Polypeptide according to aspect 139, in which said one or more    binding units are immunoglobulin sequences.

-   141. Polypeptide according to any of aspects 139 or 140, in which    said one or more other binding units are chosen from the group    consisting of domain antibodies, amino acid sequences that are    suitable for use as a domain antibody, single domain antibodies,    amino acid sequences that are suitable for use as a single domain    antibody, “dAb”'s, amino acid sequences that are suitable for use as    a dAb, or NANOBODIES.

-   142. Polypeptide according to any of aspects 139 to 141, in which    said one or more amino acid sequences of the invention are    immunoglobulin sequences.

-   143. Polypeptide according to any of aspects 139 to 142, in which    said one or more amino acid sequences of the invention are chosen    from the group consisting of domain antibodies, amino acid sequences    that are suitable for use as a domain antibody, single domain    antibodies, amino acid sequences that are suitable for use as a    single domain antibody, “dAb”'s, amino acid sequences that are    suitable for use as a dAb, or NANOBODIES.

-   144. Polypeptide according to any of aspects 139 to 143, that    comprises or essentially consists of one or more NANOBODIES    according to any of aspects 98 to 138 and in which said one or more    other binding units are NANOBODIES.

-   145. Polypeptide according to any of aspects 139 to 144, which is a    multivalent construct.

-   146. Polypeptide according to any of aspects 139 to 145, which is a    multiparatopic construct.

-   147. Polypeptide according to any of aspects 139 to 146, which is a    multispecific construct.

-   148. Polypeptide according to any of aspects 139 to 147, which has    an increased half-life, compared to the corresponding amino acid    sequence according to any of aspects 1 to 97 per se or NANOBODY    according to any of aspects 98 to 138 per se, respectively.

-   149. Polypeptide according to aspect 148, in which said one or more    other binding units provide the polypeptide with increased    half-life, compared to the corresponding amino acid sequence    according to any of aspects 1 to 97 per se or NANOBODY according to    any of aspects 98 to 138 per se, respectively.

-   150. Polypeptide according to aspect 149, in which said one or more    other binding units that provide the polypeptide with increased    half-life is chosen from the group consisting of serum proteins or    fragments thereof, binding units that can bind to serum proteins, an    Fc portion, and small proteins or peptides that can bind to serum    proteins.

-   151. Polypeptide according to aspect 149, in which said one or more    other binding units that provide the polypeptide with increased    half-life is chosen from the group consisting of human serum albumin    or fragments thereof.

-   152. Polypeptide according to aspect 149, in which said one or more    other binding units that provides the polypeptide with increased    half-life are chosen from the group consisting of binding units that    can bind to serum albumin (such as human serum albumin) or a serum    immunoglobulin (such as IgG).

-   153. Polypeptide according to aspect 149, in which said one or more    other binding units that provides the polypeptide with increased    half-life are chosen from the group consisting of domain antibodies,    amino acid sequences that are suitable for use as a domain antibody,    single domain antibodies, amino acid sequences that are suitable for    use as a single domain antibody, “dAb”'s, amino acid sequences that    are suitable for use as a dAb, or NANOBODIES that can bind to serum    albumin (such as human serum albumin) or a serum immunoglobulin    (such as IgG).

-   154. Polypeptide according to aspect 149, in which said one or more    other binding units that provides the polypeptide with increased    half-life is a NANOBODY that can bind to serum albumin (such as    human serum albumin) or a serum immunoglobulin (such as IgG).

-   155. Polypeptide according to any of aspects 148 to 154, that has a    serum half-life that is at least 1.5 times, preferably at least 2    times, such as at least 5 times, for example at least 10 times or    more than 20 times, greater than the half-life of the corresponding    amino acid sequence according to any of aspects 1 to 97 per se or    NANOBODY according to any of aspects 98 to 138 per se, respectively.

-   156. Polypeptide according to any of aspects 148 to 155, that has a    serum half-life that is increased with more than 1 hours, preferably    more than 2 hours, more preferably more than 6 hours, such as more    than 12 hours, or even more than 24, 48 or 72 hours, compared to the    corresponding amino acid sequence according to any of aspects 1 to    97 per se or NANOBODY according to any of aspects 98 to 138 per se,    respectively.

-   157. Polypeptide according to any of aspects 148 to 156, that has a    serum half-life in human of at least about 12 hours, preferably at    least 24 hours, more preferably at least 48 hours, even more    preferably at least 72 hours or more; for example, of at least 5    days (such as about 5 to 10 days), preferably at least 9 days (such    as about 9 to 14 days), more preferably at least about 10 days (such    as about 10 to 15 days), or at least about 11 days (such as about 11    to 16 days), more preferably at least about 12 days (such as about    12 to 18 days or more), or more than 14 days (such as about 14 to 19    days).

-   158. Compound or construct, that comprises or essentially consists    of one or more amino acid sequences according to any of aspects 1 to    97 and/or one or more NANOBODIES according to any of aspects 98 to    138, and optionally further comprises one or more other groups,    residues, moieties or binding units, optionally linked via one or    more linkers.

-   159. Compound or construct according to aspect 158, in which said    one or more other groups, residues, moieties or binding units are    amino acid sequences.

-   160. Compound or construct according to any of aspects 158 to 159,    in which said one or more linkers, if present, are one or more amino    acid sequences.

-   161. Compound or construct according to any of aspects 158 to 160,    in which said one or more other groups, residues, moieties or    binding units are immunoglobulin sequences.

-   162. Compound or construct according to any of aspects 158 to 161,    in which said one or more other groups, residues, moieties or    binding units are chosen from the group consisting of domain    antibodies, amino acid sequences that are suitable for use as a    domain antibody, single domain antibodies, amino acid sequences that    are suitable for use as a single domain antibody, “dAb”'s, amino    acid sequences that are suitable for use as a dAb, or NANOBODIES.

-   163. Compound or construct according to any of aspect 158 to 162, in    which said one or more amino acid sequences of the invention are    immunoglobulin sequences.

-   164. Compound or construct according to any of aspects 158 to 163,    in which said one or more amino acid sequences of the invention are    chosen from the group consisting of domain antibodies, amino acid    sequences that are suitable for use as a domain antibody, single    domain antibodies, amino acid sequences that are suitable for use as    a single domain antibody, “dAb”'s, amino acid sequences that are    suitable for use as a dAb, or NANOBODIES.

-   165. Compound or construct, that comprises or essentially consists    of one or more NANOBODIES according to any of aspects 98 to 138 and    in which said one or more other groups, residues, moieties or    binding units are NANOBODIES.

-   166. Compound or construct according to any of aspects 158 to 165,    which is a multivalent construct.

-   167. Compound or construct according to any of aspects 158 to 166,    which is a multiparatopic construct.

-   168. Compound or construct according to any of aspects 158 to 167,    which is a multispecific construct.

-   169. Compound or construct according to any of aspects 158 to 168,    which has an increased half-life, compared to the corresponding    amino acid sequence according to any of aspects 1 to 97 per se or    NANOBODY according to any of aspects 98 to 138 per se, respectively.

-   170. Compound or construct according to aspect 169, in which said    one or more other groups, residues, moieties or binding units    provide the compound or construct with increased half-life, compared    to the corresponding amino acid sequence according to any of aspects    1 to 97 per se or NANOBODY according to any of aspects 98 to 138 per    se, respectively

-   171. Compound or construct according to aspect 170, in which said    one or more other groups, residues, moieties or binding units that    provide the compound or construct with increased half-life is chosen    from the group consisting of serum proteins or fragments thereof,    binding units that can bind to serum proteins, an Fc portion, and    small proteins or peptides that can bind to serum proteins.

-   172. Compound or construct according to aspect 170, in which said    one or more other groups, residues, moieties or binding units that    provide the compound or construct with increased half-life is chosen    from the group consisting of human serum albumin or fragments    thereof.

-   173. Compound or construct according to aspect 170, in which said    one or more other groups, residues, moieties or binding units that    provides the compound or construct with increased half-life are    chosen from the group consisting of binding units that can bind to    serum albumin (such as human serum albumin) or a serum    immunoglobulin (such as IgG).

-   174. Compound or construct according to aspect 170, in which said    one or more other groups, residues, moieties or binding units that    provides the compound or construct with increased half-life are    chosen from the group consisting of domain antibodies, amino acid    sequences that are suitable for use as a domain antibody, single    domain antibodies, amino acid sequences that are suitable for use as    a single domain antibody, “dAb”'s, amino acid sequences that are    suitable for use as a dAb, or NANOBODIES that can bind to serum    albumin (such as human serum albumin) or a serum immunoglobulin    (such as IgG).

-   175. Compound or construct according to aspect 170, in which said    one or more other groups, residues, moieties or binding units that    provides the compound or construct with increased half-life is a    NANOBODY that can bind to serum albumin (such as human serum    albumin) or a serum immunoglobulin (such as IgG).

-   176. Compound or construct according to any of aspects 169 to 175,    that has a serum half-life that is at least 1.5 times, preferably at    least 2 times, such as at least 5 times, for example at least 10    times or more than 20 times, greater than the half-life of the    corresponding amino acid sequence according to any of aspects 1 to    97 per se or NANOBODY according to any of aspects 98 to 138 per se,    respectively.

-   177. Compound or construct according to any of aspects 169 to 176,    that has a serum half-life that is increased with more than 1 hours,    preferably more than 2 hours, more preferably more than 6 hours,    such as more than 12 hours, or even more than 24, 48 or 72 hours,    compared to the corresponding amino acid sequence according to any    of aspects 1 to 978 per se or NANOBODY according to any of aspects    98 to 138 per se, respectively.

-   178. Compound or construct according to any of aspects 169 to 177,    that has a serum half-life in human of at least about 12 hours,    preferably at least 24 hours, more preferably at least 48 hours,    even more preferably at least 72 hours or more; for example, of at    least 5 days (such as about 5 to 10 days), preferably at least 9    days (such as about 9 to 14 days), more preferably at least about 10    days (such as about 10 to 15 days), or at least about 11 days (such    as about 11 to 16 days), more preferably at least about 12 days    (such as about 12 to 18 days or more), or more than 14 days (such as    about 14 to 19 days).

-   179. Monovalent construct, comprising or essentially consisting of    one amino acid sequence according to any of aspects 1 to 97 and/or    one NANOBODY according to any of aspects 98 to 138.

-   180. Monovalent construct according to aspect 179, in which said    amino acid sequence of the invention is chosen from the group    consisting of domain antibodies, amino acid sequences that are    suitable for use as a domain antibody, single domain antibodies,    amino acid sequences that are suitable for use as a single domain    antibody, “dAb”'s, amino acid sequences that are suitable for use as    a dAb, or NANOBODIES.

-   181. Monovalent construct, comprising or essentially consisting of    one NANOBODY according to any of aspects 98 to 138.

-   182. Nucleic acid or nucleotide sequence, that encodes an amino acid    sequence according to any of aspects 1 to 97, a NANOBODY according    to any of aspects 98 to 138, a polypeptide according to any of    aspects 139 to 157, a compound or construct according to any of    aspects 158 to 178, or a monovalent construct according to any of    aspects 179 to 181.

-   183. Nucleic acid or nucleotide sequence according to aspect 182,    that is in the form of a genetic construct.

-   184. Host or host cell that expresses, or that under suitable    circumstances is capable of expressing, an amino acid sequence    according to any of aspects 1 to 97, a NANOBODY according to any of    aspects 98 to 138, a polypeptide according to any of aspects 139 to    157, a compound or construct according to any of aspects 158 to 178,    or a monovalent construct according to any of aspects 179 to 181;    and/or that comprises a nucleic acid or nucleotide sequence    according to aspect 182, or a genetic construct according to aspect    183.

-   185. Composition, comprising at least one amino acid sequence    according to any of aspects 1 to 97, NANOBODY according to any of    aspects 98 to 138, polypeptide according to any of aspects 139 to    157, compound or construct according to any of aspects 158 to 178,    monovalent construct according to any of aspects 179 to 181, or    nucleic acid or nucleotide sequence according to aspects 182 to 183.

-   186. Composition according to aspect 185, which is a pharmaceutical    composition.

-   187. Composition according to aspect 186, which is a pharmaceutical    composition, that further comprises at least one pharmaceutically    acceptable carrier, diluent or excipient and/or adjuvant, and that    optionally comprises one or more further pharmaceutically active    polypeptides and/or compounds.

-   188. Method for producing an amino acid sequence according to any of    aspects 1 to 97, a NANOBODY according to any of aspects 98 to 138, a    polypeptide according to any of aspects 139 to 157, a compound or    construct according to any of aspects 158 to 178, a pharmaceutical    composition according to any of aspects 186 or 187, or a monovalent    construct according to any of aspects 179 to 181, that is such that    it can be obtained by expression of a nucleic acid or nucleotide    sequence encoding the same, said method at least comprising the    steps of:    -   expressing, in a suitable host cell or host organism or in        another suitable expression system, a nucleic acid or nucleotide        sequence according to aspect 182, or a genetic construct        according to aspect 183, optionally followed by:    -   isolating and/or purifying the amino acid sequence according to        any of aspects 1 to 97, the NANOBODY according to any of aspects        98 to 138, the polypeptide according to any of aspects 139 to        157, the compound or construct according to any of aspects 158        to 178, that is such that it can be obtained by expression of a        nucleic acid or nucleotide sequence encoding the same, or the        monovalent construct according to any of aspects 179 to 181,        thus obtained.

-   189. Method for producing an amino acid sequence according to any of    aspects 1 to 97, a NANOBODY according to any of aspects 98 to 138, a    polypeptide according to any of aspects 139 to 157, a compound or    construct according to any of aspects 158 to 178, a pharmaceutical    composition according to any of aspects 186 or 187, or a monovalent    construct according to any of aspects 179 to 181, that is such that    it can be obtained by expression of a nucleic acid or nucleotide    sequence encoding the same, said method at least comprising the    steps of:    -   cultivating and/or maintaining a host or host cell according to        aspect 184 under conditions that are such that said host or host        cell expresses and/or produces at least one amino acid sequence        according to any of aspects 1 to 97, NANOBODY according to any        of aspects 98 to 138, polypeptide according to any of aspects        139 to 157, compound or construct according to any of aspects        158 to 178, that is such that it can be obtained by expression        of a nucleic acid or nucleotide sequence encoding the same, or a        monovalent construct according to any of aspects 179 to 181,    -   optionally followed by:    -   isolating and/or purifying the amino acid sequence according to        any of aspects 1 to 97, the NANOBODY according to any of aspects        98 to 138, the polypeptide according to any of aspects 139 to        157, the compound or construct according to any of aspects 158        to 178, that is such that it can be obtained by expression of a        nucleic acid or nucleotide sequence encoding the same, or the        monovalent construct according to any of aspects 179 to 181 thus        obtained.

-   190. Method for screening amino acid sequences directed against    RANK-L that comprises at least the steps of:    -   a) providing a set, collection or library of nucleic acid        sequences encoding amino acid sequences;    -   b) screening said set, collection or library of nucleic acid        sequences for nucleic acid sequences that encode an amino acid        sequence that can bind to and/or has affinity for RANK-L and        that is cross-blocked or is cross blocking a NANOBODY of the        invention, e.g. SEQ ID NO's: 560-621, or a humanized NANOBODY of        the invention, e.g. SEQ ID NO's: 730-757 and 765, or a        polypeptide or construct of the invention, e.g. SEQ ID NO's:        622-729, 759-762 and 766-773; and    -   c) isolating said nucleic acid sequence, followed by expressing        said amino acid sequence.

-   191. Method for the prevention and/or treatment of at least one bone    disease or disorder, said method comprising administering, to a    subject in need thereof, a pharmaceutically active amount of at    least one amino acid sequence according to any of aspects 1 to 97,    NANOBODY according to any of aspects 98 to 138, polypeptide    according to any of aspects 139 to 157, compound or construct    according to any of aspects 158 to 178, monovalent construct    according to any of aspects 179 to 181, or composition according to    aspect 186 or 187.

-   192. Method for the prevention and/or treatment of at least one    disease or disorder that is associated with RANK-L, with its    biological or pharmacological activity, and/or with the biological    pathways or signalling in which RANK-L is involved, said method    comprising administering, to a subject in need thereof, a    pharmaceutically active amount of at least one amino acid sequence    according to any of aspects 1 to 97, NANOBODY according to any of    aspects 98 to 138, polypeptide according to any of aspects 139 to    157, compound or construct according to any of aspects 158 to 178,    monovalent construct according to any of aspects 179 to 181, or    composition according to aspect 186 or 187.

-   193. Method for the prevention and/or treatment of at least one    disease or disorder that can be prevented and/or treated by    administering, to a subject in need thereof, an amino acid sequence    according to any of aspects 1 to 97, NANOBODY according to any of    aspects 98 to 138, polypeptide according to any of aspects 139 to    157, compound or construct according to any of aspects 158 to 178 or    a monovalent construct according to any of aspects 179 to 181, said    method comprising administering, to a subject in need thereof, a    pharmaceutically active amount of at least one amino acid sequence    according to any of aspects 1 to 97, NANOBODY according to any of    aspects 98 to 138, polypeptide according to any of aspects 139 to    157, compound or construct according to any of aspects 158 to 178,    monovalent construct according to any of aspects 179 to 181, or    composition according to aspect 186 or 187.

-   194. Method for immunotherapy, said method comprising administering,    to a subject in need thereof, a pharmaceutically active amount of at    least one amino acid sequence according to any of aspects 1 to 97,    NANOBODY according to any of aspects 98 to 138, polypeptide    according to any of aspects 139 to 157, compound or construct    according to any of aspects 158 to 178, monovalent construct    according to any of aspects 179 to 181, or composition according to    aspect 186 or 187.

-   195. Use of an amino acid sequence according to any of aspects 1 to    97, NANOBODY according to any of aspects 98 to 138, polypeptide    according to any of aspects 139 to 157, compound or construct    according to any of aspects 158 to 178 or a monovalent construct    according to any of aspects 179 to 181, in the preparation of a    pharmaceutical composition for prevention and/or treatment of at    least one bone disease or disorder; and/or for use in one or more of    the methods according to aspects 191 to 194.

-   196 Amino acid sequence according to any of aspects 1 to 97,    NANOBODY according to any of aspects 98 to 138, polypeptide    according to any of aspects 139 to 157, compound or construct    according to any of aspects 158 to 178, or a monovalent construct    according to any of aspects 179 to 181 for prevention and/or    treatment of at least one bone disease or disorder.

-   197. Part or fragment of an amino acid sequence according to any of    aspects 1 to 97, or of a NANOBODY according to any of aspects 98 to    138.

-   198. Part or fragment according to aspect 197, which is directed    against and/or can specifically bind to the RANK receptor binding    site on RANK-L.

-   199. Part or fragment according to any of aspects 197 or 198, which    is directed against and/or can specifically bind to the intersubunit    receptor-binding grooves on the RANK-L trimer.

-   200. Part or fragment according to any of aspects 197 to 199, which    modulates binding of RANKL-L to RANK.

-   201. Part or fragment according to aspect 200, which inhibits and/or    prevents binding of RANKL-L to RANK.

-   202. Part or fragment according to aspect 201, which inhibits and/or    prevents binding of RANKL-L to RANK, while not reducing and/or    inhibiting the RANK-L/OPG interaction.

-   203. Part or fragment according to any of aspects 197 to 202, which    is an antagonist of RANK-L

-   204. Part or fragment according aspect 197, which is directed    against and/or can specifically bind to the OPG binding site on    RANK-L.

-   205. Part or fragment according aspects 197 or 204, which modulates    binding of RANKL-L to OPG.

-   206. Part or fragment according to aspect 205, which inhibits the    RANK/RANK-L interaction.

-   207. Part or fragment according to aspect 206, which is an    antagonist of RANK-L.

-   208. Part or fragment according to aspect 205, which does not reduce    or inhibit the RANK/RANK-L interaction.

-   209. Part or fragment according to aspect 208, which is an agonist    of RANK-L.

-   210. Part or fragment according to aspect 197, which prevents and/or    inhibits the formation of the RANK-L trimer.

-   211. Part or fragment according to aspect 197, which prevents and/or    inhibits the differentiation and/or proliferation of osteoclasts.

-   212. Part or fragment according to aspect 197, which modulates bone    remodelling.

-   213. Part or fragment according to any of aspects 197 to 212, which    does not bind TRAIL.

-   214. Part or fragment according to any of aspects 197 to 213, which    does not bind TNF-alpha.

-   215. Part or fragment according to any of aspects 197 to 214, which    does not bind CD40 ligand.

-   216. Part or fragment according to any of aspects 197 to 215, which    does not bind related TNF family members.

-   217. Part of fragment according to any of aspects 197 to 216, that    can specifically bind to RANK-L with a dissociation constant (K_(D))    of 10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²    moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre.

-   218. Part or fragment according to any of aspects 197 to 217, that    can specifically bind to RANK-L with a rate of association    (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably    between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.

-   219. Part or fragment according to any of aspects 197 to 218, that    can specifically bind to RANK-L with a rate of dissociation (k_(off)    rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and    10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as    between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

-   220. Compound or construct, that comprises or essentially consists    of one or more parts or fragments according to any of aspects 197 to    219, and optionally further comprises one or more other groups,    residues, moieties or binding units, optionally linked via one or    more linkers.

-   221. Compound or construct according to aspect 220, in which said    one or more other groups, residues, moieties or binding units are    amino acid sequences.

-   222. Compound or construct according to any of aspects 220 or 221,    in which said one or more linkers, if present, are one or more amino    acid sequences.

-   223. Nucleic acid or nucleotide sequence, that encodes a part or    fragment according to any of aspects 197 to 219 or a compound or    construct according to aspect 222.

-   224. Composition, comprising at least one part or fragment according    to any of aspects 197 to 219, compound or construct according to any    of aspects 220 to 222, or nucleic acid or nucleotide sequence    according to aspect 223.

-   225. Derivative of an amino acid sequence according to any of    aspects 1 to 97, or of a NANOBODY according to any of aspects 98 to    138.

-   226. Derivative according to aspect 225, that can specifically bind    to RANK-L.

-   227. Derivative according to aspect 226, which is directed against    and/or can specifically bind to the RANK receptor binding site on    RANK-L.

-   228. Derivative according to any of aspects 226 or 227, which is    directed against and/or can specifically bind to the intersubunit    receptor-binding grooves on the RANK-L trimer.

-   229. Derivative according to any of aspects 226 to 228, which    modulates binding of RANKL-L to RANK.

-   230. Derivative according to aspect 229, which inhibits and/or    prevents binding of RANKL-L to RANK.

-   231. Derivative according to aspect 230, which inhibits and/or    prevents binding of RANKL-L to RANK, while not reducing and/or    inhibiting the RANK-L/OPG interaction.

-   232. Derivative according to any of aspects 226 to 231, which is an    antagonist of RANK-L

-   233. Derivative according aspect 226, which is directed against    and/or can specifically bind to the OPG binding site on RANK-L.

-   234. Derivative according aspects 226 or 233, which modulates    binding of RANKL-L to OPG.

-   235. Derivative according to aspect 234, which inhibits and/or    prevents the RANK/RANK-L interaction.

-   236. Derivative according to aspect 235, which is an antagonist of    RANK-L.

-   237. Derivative according to aspect 234, which does not reduce or    inhibit the RANK/RANK-L interaction.

-   238. Derivative according to aspect 237, which is an agonist of    RANK-L.

-   239. Derivative according to aspect 226, which prevents and/or    inhibits the formation of the RANK-L trimer.

-   240. Derivative according to aspect 226, which prevents and/or    inhibits the differentiation and/or proliferation of osteoclasts.

-   241. Derivative according to aspect 226, which modulates bone    remodelling.

-   242. Derivative according to any of aspects 226 to 241, which does    not bind TRAIL.

-   243. Derivative according to any of aspects 226 to 242, which does    not bind TNF-alpha.

-   244. Derivative according to any of aspects 226 to 243, which does    not bind CD40 ligand.

-   245. Derivative according to any of aspects 226 to 244, which does    not bind related TNF family members.

-   246. Derivative according to any of aspects 226 to 245, that can    specifically bind to RANK-L with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²    moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre.

-   247. Derivative according to any of aspects 226 to 246, that can    specifically bind to RANK-L with a rate of association (k_(on)-rate)    of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷    M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.

-   248. Derivative according to any of aspects 226 to 247, that can    specifically bind to RANK-L with a rate of dissociation (k_(off)    rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and    10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as    between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

-   249. Derivative of a compound or construct according to any of    aspects 158 to 178.

-   250. Derivative according to aspect 249, that can specifically bind    to RANK-L.

-   251. Derivative according to any of aspects 249 to 250, that can    specifically bind to RANK-L with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²    moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter.

-   252. Derivative according to any of aspects 249 to 251, that can    specifically bind to RANK-L with a rate of association (k_(on)-rate)    of between 10²M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷    M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.

-   253. Derivative according to any of aspects 249 to 252, that can    specifically bind to RANK-L with a rate of dissociation (k_(off)    rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and    10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as    between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

-   254. Derivative according to any of aspects 225 to 253, that has a    serum half-life that is at least 1.5 times, preferably at least 2    times, such as at least 5 times, for example at least 10 times or    more than 20 times, greater than the half-life of the corresponding    amino acid sequence according to any of aspects 1 to 97 per se,    NANOBODY according to any of aspects 98 to 138 per se, polypeptide    according to any of aspects 139 to 157, or compound or construct    according to any of aspects 158 to 178 per se.

-   255. Derivative according to any of aspects 225 to 254, that has a    serum half-life that is increased with more than 1 hours, preferably    more than 2 hours, more preferably more than 6 hours, such as more    than 12 hours, or even more than 24, 48 or 72 hours, compared to the    corresponding amino acid sequence according to any of aspects 1 to    97 per se or NANOBODY according to any of aspects 98 to 138 per se,    polypeptide according to any of aspects 139 to 157, or compound or    construct according to any of aspects 158 to 178 per se.

-   256. Derivative according to any of aspects 225 to 255, that has a    serum half-life in human of at least about 12 hours, preferably at    least 24 hours, more preferably at least 48 hours, even more    preferably at least 72 hours or more; for example, at least 5 days    (such as about 5 to 10 days), preferably at least 9 days (such as    about 9 to 14 days), more preferably at least about 10 days (such as    about 10 to 15 days), or at least about 11 days (such as about 11 to    16 days), more preferably at least about 12 days (such as about 12    to 18 days or more), or more than 14 days (such as about 14 to 19    days).

-   257. Derivative according to any of aspects 225 to 256, that is a    pegylated derivative.

-   258. Compound or construct, that comprises or essentially consists    of one or more derivatives according to any of aspects 225 to 257,    and optionally further comprises one or more other groups, residues,    moieties or binding units, optionally linked via one or more    linkers.

-   259. Compound or construct according to aspect 258, in which said    one or more other groups, residues, moieties or binding units are    amino acid sequences.

-   260. Compound or construct according to any of aspects 258 or 259,    in which said one or more linkers, if present, are one or more amino    acid sequences.

-   261. Nucleic acid encoding one derivative to any of aspects 225 to    257 or a compound or construct according to any of aspects 258 to    260.

-   262. Composition, comprising at least one derivative to any of    aspects 225 to 257, compound or construct according to any of    aspects 258 to 260, or nucleic acid or nucleotide sequence according    to aspects 261.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E: Results of competition ELISA with TNFα, TRAIL, CD40L andRANK-L as described in Example 3.2. FIG. 1A: binding of RANKL6 NANOBODY;FIG. 1B: binding of RANKL9 NANOBODY; FIG. 1C: binding of RANKL13NANOBODY; FIG. 1D: binding of RANKL15 NANOBODY; FIG. 1E: binding ofRANKL18 NANOBODY.

FIGS. 2A-2C: Results of cell based competition binding assay withmonovalent and trivalent anti-RANK-L NANOBODIES as described in Example4.3. FIG. 2A: inhibition with RANKL13 and RANKL130 NANOBODIES; FIG. 2B:inhibition with RANKL15 and RANKL150 NANOBODIES; FIG. 2C: inhibitionwith RANKL18 and RANKL180 NANOBODIES. Solid line: monovalent NANOBODY;Long dashed line: trivalent bispecific NANOBODY; Short dashed line:irrelevant NANOBODY.

FIGS. 3A-3C: The effects of the NANOBODIES on the differentiation ofhuman osteoclasts. The results are shown as TRACP 5b values. The groupsare: BL=Baseline (no added compounds); C=control (100 ng/ml OPG);A1=0.05 nM; A2=0.3 nM; A3=1 nM; A4=3 nM; A5=10 nM; A6=50 nM; A7=250 nM.The results of all groups were compared separately with the results ofthe baseline group using one-way ANOVA. Asterisks indicate statisticallysignificant inhibitory effects compared with baseline. All NANOBODIESinhibit dose-dependently osteoclast differentiation (***p<0.001). FIG.3A: RANKL60; FIG. 3B: RANKL130; FIG. 3C: RANK-L180.

FIGS. 4A-4B: (FIG. 4A) Serum NTx levels expressed as % change ofbaseline upon administration of NANOBODIES (values below detection limitare not included). (FIG. 4B) Average Serum NTx levels expressed as %change of baseline upon administration of small molecule Ibamdronate(IBN) or of negative control NANOBODY ALB-1.

FIG. 5: Serum BAP levels expressed as % change of baseline uponadministration of NANOBODIES.

FIG. 6: Alignment of humanized RANKL13hum5 (SEQ ID NO: 755) to the wildtype molecule RANKL13 (SEQ ID NO: 572) and to the first 97 amino acidresidues of human germline VH3-23 (SEQ ID NO: 763). Humanized aaresidues are indicated in red, while CDR1, 2 and 3 are highlighted ingreen.

FIG. 7: Analysis of the potency of RANKL13, RANKL13hum5 andRANKL13hum5_D62E in AlphaScreen assay.

FIG. 8: Binding of RANK-L130NT and RANKL 008a to immobilized RANK-L.Binding of RANK-L 130NT (light blue) and Rank-L 008a (pink) in thepresence of albumin to immobilized Rank-L.

FIG. 9: Superimposed sensorgrams of binding of RANKL18hum6. RANKL18hum6:binding of 500 nM RANKL18hum6 for 120 seconds. Mixture: binding of 500nM RANKL18hum6 for 120 seconds followed by injection of a mixturecontaining 500 nM of RANKL18hum6 and RANKL13hum5.

FIG. 10: Superimposed sensorgrams of binding of RANKL13hum5.RANKL13hum5: binding of 500 nM RANKL13hum5 for 120 seconds. Mixture:binding of 500 nM RANKL13hum5 for 120 seconds followed by injection of amixture containing 500 nM of RANKL13hum5 and RANKL18hum6.

FIG. 11: Inhibition of RANK-L induced differentiation of RAW264.7 cells.RAW264.7 cells (2000 cells/well) were incubated with a dilution seriesof RANKL008a (), RANKL003p (▪), or an irrelevant NANOBODY (▴) anddifferentiation was induced with 7.5 ng/mL RANK-L. After 4 days,tartrate-resistant acid phosphatase activity in the supernatant wasmeasured. Mean±s.e. of duplicate measurements is shown. Positivecontrols (i.e. without NANOBODY) are indicated using 0; negativecontrols (i.e. without RANK-L) are indicated using (□).

FIG. 12: Inhibition of the RANK-L interaction with RANK. A dilutionseries of RANKL008a (), RANKL003p (▴) or osteoprotegerin (▾) wasincubated with 7.5 ng/mL RANK-L and 200 ng/mL RANK-Fc. The mixtures weretransferred to a 96-well plate coated with the anti-Fc NANOBODY PMP02.Residual bound RANK-L was detected.

FIG. 13: Binding of RANKL008a to human serum albumin 96-well plates werecoated with HSA. After blocking, a dilution series of RANKL008a wasapplied. Bound RANKL008a was detected with a horseradish peroxidaselabelled bivalent NANOBODY.

FIGS. 14A-14B: Bifunctional binding of RANKL008a to HSA and RANK-L. FIG.14A) Microtiter plates were coated with neutravidin after whichbiotinylated RANK-L was bound. Wells were incubated with a dilutionseries of RANKL008a. Bound RANKL008a was detected with horseradishperoxidase labelled albumin Mean±s.e. of duplicate measurements; FIG.14B) Microtiter plates were coated with HSA after which a dilutionseries of RANKL008a was applied. Bound RANKL008a was detected withbiotinylated RANK-L followed by horseradish peroxidase labelledstreptavidin.

FIGS. 15A-15C: Serum NTx levels upon intravenous or subcutaneousadministration (3 mg/kg) of RANKL008a (FIG. 15A), RANKL001p (FIG. 15B)or RANKL003p (FIG. 15C) NANOBODIES (values below detection limit are notincluded).

FIGS. 16A-16C: Serum NTx levels upon administration (0.3 mg/kg; 0.03mg/kg) of RANKL008a (FIG. 16A), RANKL001p (FIG. 16B) or RANKL003p (FIG.16C) NANOBODIES (values below detection limit are not included).

FIG. 17: Cross-reactivity of the NANOBODIES with other TNF familymembers TNFα and TRAIL and with mouse RANK-L.

FIGS. 18A-18C: Cross-reactivity of RANKL008a with TNF family members. Atitration series of the different TNF superfamily proteins waspre-incubated with 0.08 nM RANKL008a, after which free RANKL008a wascaptured on a plate with biotinylated sRANK-L and detected with rabbitanti-VHH and anti-rabbit-HRP. The mean OD±s.e. of duplicate measurementsis shown.

FIG. 18A: competition with sRANK-L (), TNF-α (□), TRAIL (▪) and Fas-L(▾)

FIG. 18B: competition with sRANK-L (), CD40-L (▴), LTα1/β2 (□) andLTα2/β1 (∘)

FIG. 18C: competition with sRANKL () and LIGHT (□)

FIGS. 19A-19C: Inhibition of differentiation of CD14+ monocytes toosteoclasts as measured by intracellular TRACP staining. CD14+ monocyteswere isolated from PBMCs and seeded at 100.000 cells/well. Osteoclastdifferentiation was induced by addition of 50 ng/mL MCSF and 7.5 ng/mLRANKL in the absence (FIG. 19A) or presence (FIG. 19B) of 2 nMRANKL008a. As a control, wells were incubated without RANKL (FIG. 19C).After 11 days of incubation at 5% CO₂ and 37° C. in which 50% of theculture medium was replaced every 2 days by fresh medium, intracellularTRACP was stained. Representative image at 200× magnification is shown.

FIG. 20: Inhibition of differentiation of CD14+ monocytes to osteoclastsas measured by inhibition of TRACP5b activity in conditioned medium.CD14+ monocytes were isolated from PBMCs and seeded at 100.000cells/well. A dilution series of RANKL008a was added and differentiationwas induced by addition of RANKL (7.5 ng/mL) and MCSF (50 ng/mL). Cellswere cultured for 11 days and 50% of the culture medium was replacedevery 2 days by fresh medium. Differentiation was assessed by TRACP5bactivity measurement in the conditioned medium. Singlicate measurementsare shown. As a positive control, the TRACP5b activity measured in theabsence of RANKL008a (RANKL+MCSF) is indicated. As a negative control,the TRACP5b activity measured in the absence of MCSF (RANKL−MCSF) orRANKL (MCSF−RANKL) is shown.

FIG. 21: Inhibition of mRANKL binding to RANK. Microtiter plates wereimmobilized with the anti-Fc NB PMP02. CHO cells expressing human mRANKL(400.000/mL) were preincubated for 30 min at RT with RANK-Fc (500 ng/mL)in the presence of a dilution series of RANKL008a (), 13H5-CabLys3-13H5(▴) or CabLys3-Alb11-13H5 (∘) after which the mixtures were transferredto the coated wells. After 2 h of incubation at RT, adhered viable cellswere visualized.

FIG. 22: Differentiation of RAW264.7 cells by mRANKL expressed on CHOcells as measured by total TRACP activity. CHO cells recombinantlyexpressing human mRANKL were seeded in microtiter plates at a density of20.000 cells/well and fixed with paraformaldehyde. A dilution series ofRANKL008a (), 13H5-CabLys3-13H5 (▴), CabLys3-Alb11-13H5 (∘) or αOPGL-1(▪) was added after which RAW264.7 cells were added at a density of 2000cells/well. After 5 days of incubation at 5% CO₂ and 37° C., total TRACPactivity in the culture supernatant was measured (mean±s.e., n=3). TotalTRACP activity in the absence of compound or in the absence of CHO cellsis indicated as dashed lines.

FIGS. 23A-23C: Differentiation of RAW264.7 cells by mRANKL expressed onCHO cells as measured by total TRACP activity. WT CHO cells (FIG. 23A)or CHO cells recombinantly expressing human mRANKL (FIG. 23B-FIG. 23C)were seeded in microtiter plates at a density of 20.000 cells/well andfixed. RAW264.7 cells were added at a density of 2000 cells/well in theabsence (FIG. 23A-FIG. 23B) or presence (FIG. 23C) of RANKL008a (0.18nM). After 5 days of incubation at 5% CO₂ and 37° C., intracellularTRACP was stained. Representative image at 200× magnification is shown.

FIGS. 24A-24B: Individual plasma concentration-time profiles ofRANKL008a (FIG. 24A) and αOPGL-1 (FIG. 24B) following an single i.v.bolus administration at 0.3 mg/kg (RANKL008a) or 1 mg/kg (αOPGL-1) inthe female Cynomolgus monkey.

FIG. 25: Mean serum NTx-time profiles following an single i.v. bolusdose of RANKL008a at 0.3 mg/kg (circles) or of αOPGL-1 at 1 mg/kg(triangles) or PBS (squares) in the female Cynomolgus monkey.

EXAMPLES Example 1: Identification of RANK-L Blocking NANOBODIES 1.1Immunizations

Two llamas (No. 115 and No. 116) were immunized, according to standardprotocols, with 9 intramuscular injections (100 or 50 μg/dose at weeklyintervals) of alternating human RANK-L (R&D Systems, Minneapolis, Minn.,US) and mouse RANK-L (R&D Systems, Minneapolis, Minn., US) in llama No.116 and in llama No. 115 for the 5 first injections. The last fourinjections in llama 115 were only of human RANK-L. Both antigens wereformulated in Stimune (Cedi-Diagnostics B.V., Lelystad, TheNetherlands). At week 3, sera were collected to define antibody titersagainst human and mouse RANK-L by ELISA. In short, 96-well Maxisorpplates (Nunc, Wiesbaden, Germany) were coated with human or mouseRANK-L. After blocking and adding diluted sera samples, the presence ofanti-RANK-L NANOBODIES was demonstrated by using HRP (horseradishperoxidase) conjugated goat anti-llama immunoglobulin (BethylLaboratories Inc., Montgomery, Tex. USA) and a subsequent enzymaticreaction in the presence of the substrate TMB(3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, Ill., USA).OD_(450nm) exceeded 1 for human and mouse RANK-L in both animals.

1.2 Library Construction

Peripheral blood mononuclear cells were prepared from the serum samplesusing Ficoll-Hypaque according to the manufacturer's instructions. Next,total RNA was extracted from these cells and used as starting materialfor RT-PCR to amplify NANOBODY encoding gene fragments. These fragmentswere cloned into a house made phagemid vector. Phage was preparedaccording to standard methods (see for example the prior art andapplications filed by applicant cited herein) and stored after filtersterilization at 4° C. for further use.

1.3 Selections

Phage libraries obtained from llamas No. 115 and No. 116 were used fordifferent selection strategies.

In a first selection, biotinylated hRANK-L (expressed in mouse myelomaNSO cell line) (R&D Systems, Minneapolis, US) at 1, 0.1, 0.01 μg/ml wascaptured on a neutravidin coated solid phase. Following incubation withthe phage libraries and extensive washing, bound phage was aspecificallyeluted with trypsin (1 mg/ml).

In a second selection, soluble biotinylated hRANK-L (R&D Systems,Minneapolis, US) (1 nM, 100 pM, 10 pM) was incubated with the phagelibraries. After extensive washing, the biotinylated hRANK-L wascaptured on a neutravidin coated solid phase. Bound phage wasspecifically eluted with trypsin (1 mg/ml)

In a third selection hRANK-L (expressed in E. Coli) (Peprotech, London,UK) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany)at 1, 0.1, 0.01 μg/ml. Following incubation with the phage libraries andextensive washing, bound phage was aspecifically eluted with trypsin (1mg/ml).

In all selections, enrichment was observed. The output from theselections was recloned as a pool into a house made expression vector.Colonies were picked and grown in 96 deep well plates (1 ml volume) andinduced by adding IPTG for NANOBODY expression. Periplasmic extracts(volume: ˜80 μl) were prepared according to standard methods (see forexample the prior art and applications filed by applicant cited herein).

1.4 Screening for RANK-L Blocking NANOBODIES in Alphascreen Assay

The periplasmic extracts were screened in a RANK Alphascreen assay toevaluate the blocking capacity of the expressed NANOBODIES. This assayrelies on the use of Donor and Acceptor beads which can be conjugated tobiological molecules. When a biological interaction between moleculesbrings the beads into proximity, excited singlet oxygen molecules thatare produced upon laser excitation at 680 nm by a photosensitizer in theDonor bead, diffuse across to react with a chemiluminiscer in theacceptor bead that further activates fluorophores which subsequentlyemit light at 520-620 nm. If the NANOBODY inhibits binding of RANK-L toRANK, fluorescent output will decrease, and the amount of NANOBODYpresent will be inversely related to the amount of fluorescence.

Human RANK-L was biotinylated using biotin (Sigma, St Louis, Mo., US)and biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester sodiumsalt (Sigma, St Louis, Mo., US). RANK-huFc chimera (Alexis,Biochemicals, Lausen, Switzerland) was coupled to acceptor beadsaccording to manufacturer instructions (Perkin Elmer, Waltham, Mass.,US). To evaluate the neutralizing capacity of anti-hRANK-L NANOBODIES,dilution series of the periplasmic extracts were pre-incubated withbiotinylated human RANK-L. To this mixture, the acceptor beads and thestreptavidin donor beads were added and further incubated for 1 hour atroom temperature. Fluorescence was measured by reading plates on theEnVision Multilabel Plate Reader (Perkin Elmer) using an excitationwavelength of 680 nm and an emission wavelength of 520 nm Decrease influorescence signal indicates that the binding of biotinylated RANK-L tothe RANK receptor is blocked by the NANOBODY expressed in theperiplasmic extract.

From this screening inhibiting NANOBODIES were selected and sequenced.Sequencing analysis revealed 12 unique NANOBODIES and 10 NANOBODYfamilies. The corresponding sequences are depicted in Table C-1 andTable B-1. The sequence of non-inhibitory NANOBODIES is shown in TableB-2.

Example 2: Characterization of 21 RANK-L Blocking NANOBODIES inAlphascreen Assay and ELISA 2.1 NANOBODY Expression and Purification

20 inhibitory NANOBODIES selected from the screening described inexample 1 were further purified and characterised. Selected NANOBODIESwere expressed in E. coli as c-myc, His6-tagged proteins in a culturevolume of 50 mL. Expression was induced by addition of 1 mM IPTG andallowed to continue for 4 h at 37° C. After spinning the cell cultures,periplasmic extracts were prepared by freeze-thawing the pellets. Theseextracts were used as starting material for immobilized metal affinitychromatography (IMAC). NANOBODIES were eluted from the column with 150mM imidazole and subsequently dialyzed against PBS.

2.2 NANOBODIES Bind to Soluble hRANK-L Coated on Maxisorp 96-Well Plates

First, biotinylated hRANK-L (200 ng/ml) was captured on neutravidincoated 96-well plates. Therefore 2 μg/ml neutravidin was coatedovernight at 4° C. The plates were washed 5 times with 300 μl PBS andthen blocked with 300 μl 1% casein/PBS for 2 hours at room temperature.After blocking biotinylated RANK-L (200 ng/ml) was added to the wellsand incubated for 1 hour at room temperature. After extensive washingwith PBS, varying concentrations of NANOBODIES starting from 500 nM to160 pM diluted in 1% casein/PBS were added to the wells and the plateswere incubated for 1 hour at room temperature. Bound NANOBODIES weredetected by subsequent incubations of a primary mouse anti-myc antibodyand a secondary anti mouse-HRP conjugate (DAKO, Glostrup, Denmark) andusing TMB-H2O2 (Pierce, Rockford, Ill., USA) substrate cocktail. Thereaction was stopped with H₂SO₄ and the OD was read at 450 nm. AllNANOBODIES bound to hRANK-L coated on plates in a dose-dependent way.Calculated ED50 values are shown in Table C-2.

The same panel of NANOBODIES was also analysed for binding to murineRANK-L in a similar set-up as described for human RANK-L (see above).Only NANOBODY RANKL3 could bind to murine RANK-L in a dose-dependent waywith an ED50 of approximately 600 pM.

2.3 NANOBODIES Block the Binding of RANK-L to its Cognate Receptor RANKin Alpha Screen

Human RANK-L was biotinylated using biotin (Sigma, St Louis, Mo., US)and biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester sodiumsalt (Sigma, St Louis, Mo., US). RANK-huFc chimera (1 nM) (Alexis,Biochemicals, Lausen, Switzerland) were coupled to acceptor beadsaccording to manufacturer instructions (Perkin Elmer, Waltham, Mass.,US).

A dilution series of anti-RANK-L NANOBODIES starting from 50 nM up to 1pM was pre-incubated with 100 pM biotinylated RANK-L during 30 minutesat RT. To this mixture, the RANK acceptor beads and the streptavidindonor beads were added and further incubated for 1 hour at roomtemperature. Fluorescence was measured by reading plates on the EnVisionMultilabel Plate Reader (Perkin Elmer) using an excitation wavelength of680 nm and an emission wavelength of 520 nm.

Preincubation of all NANOBODIES with biotinylated RANK-L reducedfluorescence intensity at 520 nm, demonstrating that the NANOBODIES caneffectively inhibit RANK-L binding to RANK in a dose-dependent manner.The calculated IC50 values are shown in Table C-3, and vary from 137 pMfor RANKL9 up to 3530 pM for RANKL23.

Example 3: Binding Specificity of RANKL 6, 9, 13, 15, 18 3.1 RANKL 6, 9,13, 15, 18 Bind Specifically to Cell Membrane Expressed Human andCynomolgus RANK-L

RANK-L of human and cynomolgus monkey was expressed in Human embryonickidney cells (HEK293T; Wullaert et al. 2007, J. Biol. Chem. 282: 81-90)as the full length, membrane-bound protein. Binding of the NANOBODIES tothe cell surface expressed RANK-L was assessed by FACS analysis of cellsas described below.

Human embryonic kidney cells (HEK293T) were grown in Dulbecco's modifiedEagles's medium supplemented with 10% fetal bovine serum (FBS), 2 nML-glutamine, 0.4 mM sodium pyruvate. HEK293T cells were transientlytransfected with the plasmid expressing full length human or full lengthcynomolgus RANK-L using FUGENE6® (nonliposomal transfection reagent) andOptimem (Roche Molecular Biochemicals, Indianapolis, Ind., US) accordingto manufacturer's instructions. After 48 hours, transfected cells weresubjected to FACS analysis. Transfectants were seeded in 96-well platesat a final concentration of 5E+06/ml in FACS buffer (PBS+10% FBS) andincubated with varying concentrations of NANOBODY (2 nM, 400 pM, 80 pM,16 pM, 3.2 pM for hRANK-L transfectants; 2 nM, 400 pM for cynomolgusRANK-L transfectants) for 1 hour at 4° C. Then cells were washed threetimes with FACS buffer. Bound NANOBODIES were detected by subsequentincubations of a primary mouse anti Myc antibody (30 min at 4° C. (2μg/ml)) and a second incubation with goat anti mouse-Phycoerythrin (PE)(Jackson Laboratories) for another 30 min at 4° C. Finally, afterwashing, the cells were resuspended in PBS+10% FBS+TO-PRO®-3 (5 nM)(Molecular Probes®, Invitrogen, Merelbeke, Belgium) and cell surfacefluorescence expressed as mean PE fluorescence, was measured using flowcytometry. All NANOBODIES showed dose-dependent binding to both humanRANK-L and cynomolgus RANK-L expressed on HEK293T cells at allconcentrations tested (Table C-4). Binding was specific since additionof 40 nM soluble human RANK-L to the incubation mixture of 400 pMNANOBODY with the HEK293T transfectants abrogated binding of therespective NANOBODY to the cells (data not shown).

3.2 RANKL 6, 9, 13, 15, 18 do not Bind to TNF Family Members TNFα, CD40Ligand (CD40L) or TRAIL

Human OPG has been reported to display weak binding to tumor necrosisfactor-related apoptosis inducing ligand (TRAIL). The RANK-L amino acidsequence shows 34% similarity with that of TRAIL.

In a competition ELISA it was shown that the anti-RANK-L NANOBODIES donot bind to TNF family members TNFα, TRAIL and CD40L. RANK-L was coatedon 96-well plates as described in example 2. NANOBODIES RANKL 6, 9, 13,15 and 18 (1 nM) were preincubated with varying concentrations ofRANK-L, TNFα, CD40L or TRAIL (approximately 100 nM down to 40 pM) beforethey were added to the plates. Binding of the NANOBODIES to the RANK-Lcoated plates was only inhibited by exogenously added RANK-L and was notaffected by the addition of the other ligands (FIGS. 1A-1E).

Example 4: Neutralizing Activity of Trivalent Bispecific Anti RANKLNANOBODIES Versus Monovalent Counterparts 4.1 Construction andExpression of Trivalent Bispecific Anti-RANK-L NANOBODIES

RANKL3, RANKL6, RANKL9, RANKL13, RANKL15 and RANKL18 were also expressedas trivalent bispecific anti-RANK-L NANOBODIES. The trivalent molecules(e.g. RANKL6-ALB1-RANKL6) comprise of two building blocks correspondingto anti-RANK-L NANOBODIES with in the middle a third building blockcorresponding to an anti Human Serum Albumin (HSA) NANOBODY buildingblock (ALB-1; SEQ ID NO: 790). The individual building blocks were fusedby a Gly/Ser (GGGGSGGGS; SEQ ID NO: 792) linker. The sequences of thesetrivalent bispecific anti-RANK-L NANOBODIES are shown in Table B-3.These constructs were expressed in E. coli as c-myc, His6-taggedproteins and subsequently purified from the culture medium byimmobilized metal affinity chromatography (IMAC) and size exclusionchromatography (SEC).

4.2 Inhibition by Trivalent NANOBODIES of RANK-L Binding to RANK inAlpha-Screen

5 trivalent NANOBODIES (RANKL30: RANKL3-ALB1-RANKL3; RANKL60:RANKL6-ALB1-RANKL6, RANKL90: RANKL9-ALB1-RANKL9, RANKL130:RANKL13-ALB1-RANKL13, RANKL150: RANKL15-ALB1-RANKL15, RANKL180:RANKL18-ALB1-RANKL18) were compared to their monovalent counterparts inthe Alpha-Screen assay to evaluate whether they can also block RANK-Lbinding to its cognate receptor.

AlphaScreen was performed as described in Example 2. As shown in TableC-5, all trivalent NANOBODIES blocked RANK-L binding to the RANKreceptor in a dose dependent way with increased potency as compared tothe corresponding monovalent molecules.

4 3 Inhibition by Anti-RANK-L NANOBODIES of RANK-L Binding to RANKExpressed on HEK293T Cell Membranes

HEK293T cells were transiently transfected with the plasmid expressingfull length RANK using FUGENE6 as described in example 3. After 24 hoursaliquots of 60 μl (7.5×10³ cells) were plated into FMAT system 384-wellplates (PE Biosystems, CA, US) and allowed to adhere for 24 h. Afterovernight adherence, culture supernatant was removed by gently tappingthe plate. To initiate the competitive screen, 20 μl ALEXA⁶⁴⁷-labeledhuman RANK-L (200 pM final concentration) diluted in PBS+10% BSA (FMATbuffer) and 20 μl of a dilution series (200 nM down to 0.075 pM) of thedifferent monovalent and trivalent NANOBODIES were added to thecell-containing FMAT system 384-well plates (PE Biosystems, CA). Theplates were scanned after 10 hours of incubation. Cell surfacefluorescence was measured by 8200 Cellular Detection System (AppliedBiosystems, Foster City, Calif., US) which is a fluorescencemacro-confocal, biological binding event analyzer that enablesmix-and-read assays with live cells.

Table C-6 and FIGS. 2A-2C show that NANOBODIES blocked binding of RANK-Lto its receptor in a dose dependent way and that the trivalent formatsshow increased potency over the monovalent molecules.

4.4 Inhibition by Anti-RANK-L NANOBODIES of RANK-L Induced NF-KBActivation in HEK293T Cells

RANK-L stimulated osteoclastogenesis is associated with NF-κBactivation. Most likely, RANK-L activates the most common dimer, p50/p65(Wei et al. 2001, Endocrinology 142(3): 1290-1295). The important roleof NF-kB p50 and p52 molecules in osteoclastogenesis has been shown byXing et al. (2002, J Bone Miner. Res. 17(7): 1200-1210) reporting thatNF-κB p50 and p52 are essential for RANK-expressing osteoclastprecursors to differentiate into TRAP+ osteoclasts in response toRANK-L. Moreover p50 and p52 double knock out mice reportedly developsevere osteopetrosis due to the inability to generate mature osteoclasts(Franzoso et al. 1998, Genes & Development 11: 3482-3496). All together,RANK-L induced NF-κB activation is important for formation of matureosteoclasts.

To evaluate the effect of NANOBODIES on RANK-L induced NF-κB activation,HEK293T cells were transiently transfected as described in example 3with a NF-κB reporter gene plasmid and a plasmid encodingβ-galactosidase, the latter being used to correct for transfectionefficiency. After 24 h, the cells were seeded in 96-well plates. Another24 h later, cells were left untreated or were incubated in the presenceof a constant amount of human RANK-L (300 ng/ml) and varying amounts ofthe indicated anti-RANK-L NANOBODIES (200 nM down to approximately 10pM). After 6 hours cells were lysed; luciferase (Luc) andβ-galactosidase activity were assayed using the Dual-light kit(Tropix/Applied Biosystems, Foster City, Calif., US) according tomanufacturer's instructions. Luc values were normalized forβ-galactosidase values to correct for differences in transfectionefficiency. As shown in Table C-7, all anti-RANK-L NANOBODIES inhibitRANK-L induced NF-κB activation in a dose-dependent way. Calculated IC50values indicate that trivalent NANOBODIES are more potent then thecorresponding monovalent molecules.

Example 5: Inhibition of Osteoclast Formation by NANOBODIES

The effects of the three NANOBODIES RANKL60, RANKL130 and RANKL180 ondifferentiation of human osteoclasts in vitro were investigated.

The method of osteoclast culture on bone slices was originally describedby Boyde and co-workers (1984; Br. Dent J. 156: 216-220) and by Chambersand Horton (1984; J. Pathol. 144: 295-6). Originally, the number ofosteoclasts was determined by calculating the number oftartrate-resistant acid phosphatase (TRACP)-positive multinuclear cellsunder a microscope. Later, it was demonstrated that secreted TRACP 5bactivity reflects the number of osteoclasts in mouse osteoclast cultures(Alatalo et al., 2000; Clin. Chem. 46: 1751-4). While secreted TRACP 5bactivity correlated strongly with the number of osteoclasts, TRACP 5bwas not secreted by TRACP-positive mononuclear osteoclast precursorcells before they had differentiated into mature multinuclearosteoclasts. Therefore, secreted TRACP 5b is a reliable marker of thenumber of mature multinuclear osteoclasts.

A human osteoclast differentiation assay was set up in which CD34+osteoclast precursor cells derived from human bone marrow were culturedfor 7 days in the presence of appropriate growth factors, includingM-CSF and RANK-ligand, allowing them to differentiate into maturebone-resorbing osteoclasts (Rissanen et al., 2005; Circulation 112:3937-46).

Human bone marrow-derived CD34+ stem cells were suspended to culturemedium and allowed to attach to bovine bone slices in 96-well tissueculture plates. The culture medium contained appropriate amounts ofimportant growth factors favoring osteoclast differentiation, includingM-CSF and RANK-ligand. The cells were incubated in a CO2 incubator inhumidified atmosphere of 95% air and 5% carbon dioxide at 37° C. At day7 when osteoclast differentiation was completed, tartrate-resistant acidphosphatase isoform 5b activity (TRACP 5b) was measured from the culturemedium as an index of the number of osteoclasts formed, using theBONETRAP® assay (test for the quantitative determination of the activeisoform 5b of the tartrate-resistant acid phosphatase (TRACP); IDS Ltd,Boldon, UK) and VICTOR2™ Multilabel Counter (PerkinElmer, Waltham,Mass., USA).

Seven different concentrations of each NANOBODY were tested in thisstudy, ranging from 0.05 nM to 250 nM. A baseline group withoutNANOBODIES and a control group with a reference molecule were includedin each cell culture. Osteoprotegerin, an inhibitor ofosteoclastogenesis, was used as a reference molecule to demonstrate thatthe test system can detect inhibition of osteoclast differentiation.

Baseline TRACP 5b values were high and the reference inhibitor OPGinhibited significantly osteoclast differentiation, which indicates thatthe assay was performed successfully and the results obtained reliable(FIGS. 3A-3C). All three NANOBODIES inhibited dose-dependently humanosteoclast differentiation. All compounds showed a statisticallysignificant inhibition with 3.0 nM and higher concentrations. Theinhibition profiles were similar for all three NANOBODIES.

Example 6: Identification of Residues on RANK-L Involved in theInteraction with the NANOBODIES 6.1 Construction of Human/Mouse Hybrids

In order to identify the binding sites of the NANOBODIES on the RANK-Lmolecule, the human RANK-L-specific binding of the NANOBODIES wasexploited. Except for NANOBODY RANKL3, the obtained NANOBODIES did notinteract with mouse RANK-L. In order to identify residues involved inthe species-specific interaction on human RANK-L, three human/mousehybrids were designed containing human/mouse substitutions in AA′ and CDloops as follows:

AA′ Loop:

human/mouse hybrid 1: T¹⁷⁴D¹⁷⁵ were substituted with A and Srespectively

CD Loop:

human/mouse hybrid 2: D²³¹L²³² were substituted with S and Vrespectivelyhuman/mouse hybrid 3: A²³³ TE ²³⁵ were substituted with P and TDrespectively

The human/mouse hybrids were generated by overlap PCR using the primersshowed in Table C-8 Amplicons were subsequently cloned as Xho1/Xba1restriction fragments in an expression vector, pClneo (Promega, Madison,Wis.).

6.2 Binding of NANOBODIES to RANK-L Human/Mouse Hybrids

HEK293T cells were transiently transfected with expression vectorsencoding hRANK-L or the human/mouse hybrids. Binding of the NANOBODIES(varying concentrations: 250 nM, 50 nM, 10 nM, 2 nM, 0.4 nM, 0.08 nM, 16pM) to the cell surface expressed RANK-L was assessed by FACS analysisof cells as described above. An overview of the binding results ispresented in Table C-9.

Binding of the human/mouse cross-reactive RANKL3 NANOBODY served as acontrol for expression and correct folding of the different hybridmolecules. As shown in Table C-9, RANKL3 binds to the three differenthybrid molecules.

The binding results with the human-specific NANOBODIES can be summarizedas follows:

-   -   Loop AA′ is not involved in the species-specific interaction        with the NANOBODIES.    -   Loop CD is crucial for the interaction with the NANOBODIES.    -   NANOBODIES can be subdivided in different classes based on the        observed differences in their interaction with the CD Loop.        -   RANKL13, RANKL15: Mutation of the N-terminal part and            C-terminal part of the CD-loop abrogates the interaction.        -   RANKL9 and RANKL18: Mutation of the C-terminal part of the            CD-loop abrogates the interaction. N-terminal part of the            CD-loop is not involved in the interaction.        -   RANKL6: Mutation of the N-terminal part of the CD-loop            abrogates the interaction. C-terminal part of the CD-loop is            not involved in the interaction.

Example 7: Pharmacokinetics and Pharmacodynamics in Cynomolgus Monkeys

Five males and five female cynomolgus monkeys were assigned to 5 groups,each group consisting of one male and one female. Male individuals wereaged between 39 and 42 months, while females had reached the age of 33to 44 months. The animal's initial body weight varied between 2.5 and2.7 kg for the male individuals, and 2.2 and 2.6 kg for the femaleindividuals.

NANOBODIES RANKL130 (SEQ ID NO: 715) and RANKL180 (SEQ ID NO: 729) weretested in addition to NANOBODY RANKL131 (SEQ ID NO: 643). RANKL131corresponds to a bivalent NANOBODY composed solely of twolinker-interconnected RANKL13 building blocks. RANKL131 was included tofully assess the impact of the albumin-binding format on both PK and PD.ALB-1 (SEQ ID NO: 790) was included as a negative control and smallmolecule Ibandronate (LKT Laboratories, Inc, St. Paul, Minn.) served asa positive control. Animals were dosed as described in Table C-10.

Serum samples were taken for determination of the NANOBODY levels,antibody analysis, and analysis of the bone turnover markers serumN-telopeptide (serum N-Tx) and BAP (bone-specific alkaline phosphatase).Urine was also collected for analysis of N-telopeptide (urine N-Tx) andcreatinine.

7.1 Pharmacokinetics

Concentrations of NANOBODIES RANKL130, RANKL180 and RANKL131 weredetermined in plasma as follows: 96-well microtiter plates (Maxisorp,Nunc, Wiesbaden, Germany) were coated overnight at 4° C. with 100 μL,neutravidin (2 μg/mL, Pierce, Rockford, Ill.). Wells were aspirated andblocked for 30 min at RT with 300 μL, SuperBlock®T20 PBS (Pierce,Rockford, Ill.). After 3 washing steps with PBS-0.05% TWEEN20(Polysorbate 20), biotinylated RANKL (0.5 μg/mL in PBS-0.1% casein-0.05%TWEEN20) was captured by incubating 100 μL, for 1 hr at RT while shakingat 600 rpm. After this incubation step, wells were washed 3 times withPBS-0.05% TWEEN20. The standards, QC and predilutions of the testsamples were prepared in a non-coated (polypropylene) plate in 100%cynomolgus monkey plasma and incubated for 30 min at RT while shaking at600 rpm. A 1/10 dilution of the samples and standards in PBS-0.1%casein-0.05% TWEEN20 (final concentration of cynomolgus monkey plasma is10%) was transferred to the coated plate and incubated for 1 hr at RTwhile shaking at 600 rpm. After three washing steps with PBS-0.05%TWEEN20, the plates were incubated with an in-house purified rabbitanti-NANOBODY polyclonal antibody (1 μg/mL in PBS-0.1% casein-0.05%TWEEN20) for 1 hr at RT while shaking at 600 rpm. After 3 washing stepswith PBS-0.05% TWEEN20, 100 μl horse radish peroxidase (HRP) labeledpolyclonal goat anti-rabbit (1/5000 in PBS-0.1% casein-0.05% TWEEN20,DakoCytomation, Glostrup, Denmark) was incubated for 1 hr at RT whileshaking at 600 rpm. Visualization was performed covered from light for10 min with 100 μL, enhanced soluble 3,3′,5,5′-tetramethylbenzidine(esTMB, SDT, Brussels, Belgium), 1/3 diluted in substrate buffer. Thissubstrate buffer was a composition of 60% Na₂HPO₂ (100 mM) and 40%citric acid (100 mM). After 10 min, the colouring reaction was stoppedwith 100 μL, 1N HCl. The absorbance was determined at 450 nm after a 10sec shake in the Tecan ELISA reader, and corrected for backgroundabsorbance at 620 nm Concentration in each sample was determined basedon a sigmoid standard curve.

Profiles for RANKL130 and RANKL180 plasma concentrations seemed todecline in a triphasic manner. In the first 2.5 days postadministration, there was an initial short disposition phase (apparentalpha phase t½ ≈0.5 days), followed by a dominant slower secondary phase(apparent beta phase) and a short final phase (apparent gamma phase)characterized by a change in terminal slope.

Individual plasma concentration-time profiles of all individualsinjected with RANKL130 and RANKL180 were subjected to anon-compartmental pharmacokinetic analysis (NCA) using thepre-programmed Model 201 within WinNonlin Professional Software Version5.1 (Pharsight Corporation, Mountain View Calif., USA). Individualplasma concentration-time profiles of all animals injected with RANKL131were analyzed using the pre-programmed Model 202. The area under theplasma concentration-curve (AUC) and derived PK-parameters werecalculated by means of the linear-up/log down trapezoidal rule. Anoverview of the calculated pharmacokinetic parameters is presented inTable C-11 and Table C-12.

Significant antibody titres to NANOBODIES are detected in four out ofsix animals from the RANKL130, RANKL131 and RANKL180 cohorts. Theincidence was not NANOBODY-dependent since the four animals thatdeveloped antibodies originate from three different cohorts (cynolm,cyno3m, cyno4f, cyno6f).

7.2 Pharmacodynamics

The discovery of cross-linked N-telopeptides of type I collagen (NTx)has provided a specific biochemical marker of human bone resorption.Generation of the NTx molecule is mediated by osteoclasts on bone andfound in urine and serum as a stable end-product of degradation.

Changes in bone resorption induced by the NANOBODIES were assessed byassaying serum NTx and urine NTx using immunoassays according tomanufacturer's instructions (Osteomark® NTx serum, Osteomark® NTx urine,Wampole Laboratories).

RANKL130 and RANKL180 caused a rapid decrease in serum NTx levels (FIGS.4A-4B). Maximal inhibition in these two test groups lasted up to atleast 40 days. The serum NTx levels started to return to baseline aroundday 40 for cyno 1m and cyno 6f. In cyno 2f and 5m, the return tobaseline initiated on day 45 and day 52, respectively. The lessfavourable profiles seen for cyno 1m and cyno 6f can be explained by theobserved immunogenicity in these animals.

The suppression of serum NTx profiles induced by RANKL131 in cyno 3m andcyno 4f was transient, as the levels return to baseline by day 8.

The small molecule drug Ibandronate, which was administered oncemonthly, induced an overall inhibition of approximately 50% compared tobaseline levels.

Urine NTx levels were measured up to day 16 for the RANKL130, RANKL131and RANKL180 cohorts. Urine NTx showed similar trends to those of serumNTx.

Changes in bone formation were assessed by assaying bone-specificalkaline phosphatase (BAP) activity in serum as a quantitative measureand indicator of osteoblastic activity using the Metra® BAP immunoassay.The assay was performed according to manufacturer's instructions.RANKL130 and RANKL180 induced suppression of BAP activity (FIG. 5).

Example 8: Humanization of NANOBODIES

Humanized versions of the wild type NANOBODIES were assembled fromoligonucleotides using a PCR overlap extension method. The sequences ofdifferent possible variants of RANKL6, 9, 13, 15 and 18 that wereevaluated for their binding capacity and neutralizing activity inAlphascreen and in the biochemical and cellular assays as described inexamples 2, 3 and 4 are shown in Table B-5.

RANKL13

The amino acid sequence of anti-RANKL NANOBODY RANKL13 (SEQ ID NO: 572)was blasted to the human germline V_(H) sequence database using anin-house sequence query/alignment tool (FIG. 6). Human germline VH3-23(DP-47; SEQ ID NO: 763-764) showed the closest related sequence.NANOBODY RANKL13 shows 80 identical and 7 extra conservative amino acidsubstitutions over the first 97 amino acid residues of human germlineVH3-23. 8 amino acid residues (indicated in red) were substituted forhumanization purposes to make RANKL13hum5 (SEQ ID NO: 755).

In the humanization process of RANKL13, five RANKL13 versions(RANKL13basic, RANKL13hum1, RANKL13hum2, RANL13hum3 and RANKL13hum4)were constructed. RANKL13basic contains 4 substitutions: A14P, E44G,V78L and Q108L. In addition to these changes, additional substitutionshave been introduced in the hum1-4 versions: RANKL13hum1: R27F;RANKL13hum2: R305; RANKL13hum3: A49S; RANKL13hum4: S91Y. All versionswere tested in AlphaScreen assay and binding to RANK-L was analysed bysurface plasmon resonance.

All versions were tested in AlphaScreen assay. Calculated IC50 values inAlphaScreen indicate that the introduced mutations did not affect thepotencies of the humanized RANKL13 versions when compared to the wildtype RANKL13 (Table C-13).

Binding kinetics of the humanized versions of NANOBODY RANKL13 were alsoanalysed by Surface Plasmon Resonance (BIACORE 3000). Human solubleRANK-L was covalently bound to CM5 sensor chips surface via aminecoupling using EDC/NHS for activation and HCl for deactivation. NANOBODYbinding was assessed at one concentration (100 nM). Each NANOBODY wasinjected for 4 minutes at a flow rate of 45 μl/min to allow binding tochip-bound antigen. Next, binding buffer without NANOBODY was sent overthe chip at the same flow rate to allow spontaneous dissociation ofbound NANOBODY. From the sensorgrams obtained for the differentNANOBODIES k_(off)-values (k_(d)) were calculated and are indicated inTable C-13. Association rate constants (k_(on) or k_(a)), and hence alsoK_(D) values, were only indicative as only 1 concentration of NANOBODYwas used to fit the binding model. As shown in Table C-13, all humanizedNANOBODIES showed comparable dissociation rate constants/off ratescompared to the wild type NANOBODY RANKL13.

All together, AlphaScreen data and BIACORE analysis did not indicatesignificant effects on binding or potency of the introduced mutations inthe respective humanized versions. In a final humanized version,RANKL13hum5, all humanizing mutations were combined. As shown in TableC-13, RANKL13hum5 shows similar potency and binding affinity as the wildtype RANKL13.

RANKL18

In the humanization process of RANKL18, six RANKL18 versions(RANKL18basic, RANKL18hum1, RANKL18hum2, RANL18hum3, RANKL18hum4 andRANKL18hum5) were constructed. RANKL18basic contains 4 substitutions:A14P, E44G, V78L and Q108L. In addition to these changes additionalsubstitutions were introduced in the hum1-5 versions: RANKL18hum1: R27F;RANKL18hum2: G49S; RANKL18hum3: G60A; RANKL18hum4: P77T and V78L;RANKL18hum5: G94A. All versions were tested in AlphaScreen and BIACORE(Table C-13).

Calculated IC50 values in AlphaScreen indicated that the introducedmutations in RANKL18basic, RANKL18hum3 and RANKL18hum5 did not affectthe potencies of these versions compared to the wild type RANKL18 (TableC-13). Humanizing mutations in RANKL18hum1 and RANKL18hum2 influencedmoderately the potency while the double mutation in RANKL18hum4completely abrogated the potency of the NANOBODY. Based on theseresults, two additional mutants, RANKL18hum6 and RANKL18hum7 wereconstructed and analysed. RANKL18hum6 combines humanizing mutations inRANKL18basic, RANKL18hum3 and RANKL18hum5. RANKL18hum7 includes allmutations of RANKL18hum6 together with V78L substitution. Table C-13shows that RANKL18hum6 displays similar potency and binding affinity asthe wild type RANKL18. RANKL18hum7 is slightly less potent and shows aslightly reduced binding affinity to RANK-L.

Example 9: Analysis of RANKL13hum5 D62E Mutant

Analysis of the primary sequence of RANKL13hum5 identified D62 as apotential site for isomerisation and hence as a potential source forchemical instability of the molecule. To test this possibility, astability assay was performed with the RANKL13hum5 molecule and a mutantin which the potential isomerisation site is replaced by a glutamic acidresidue (E), RANKL13hum5_D62E (SEQ ID NO: 756).

The D62E mutation in RANKL13hum5 was introduced by overlap PCR usingprimers including the mutation: RevRANKL13hum5D62(CCTCCCTTTGACGGATTCCGCGTAATACGT; SEQ ID NO: 797) and FwRANKL13hum5D62E(ACGTATTACGCGGATTCCGTCAAAGGGAGG; SEQ ID NO: 798). Both cDNAs encodingRANKL13hum5 or RANKL13hum5_D62E were cloned as SfiI/BstEII fragments inan expression vector derived from pUC119 which contained the LacZpromoter, a resistance gene for ampicillin or carbenicillin, amulticloning site and the gen3 leader sequence. In frame with theNANOBODY coding sequence, the vector coded for two stop codons at the 3′end of the NANOBODY.

For production, RANKL13hum5 and RANKL13hum5_D62E constructs wereinoculated in 50 ml TB/0.1% glucose/Kanamycin and the suspensionincubated overnight at 37° C. 5×400 ml medium was inoculated with 1/100of the obtained overnight preculture. Cultures were further incubated at37° C., 250 rpm until OD600>5. The cultures were induced with 1 mM IPTGand further kept incubating for 4 hours at 37° C. 250 rpm. The cultureswere centrifuged for 20 minutes at 4500 rpm and afterward thesupernatant was discarded. The pellets were stored at −20° C.

For purification, pellets were thawed and re-suspended in 20 mL d-PBSand incubated for 1 hour at 4° C. Then, suspensions were centrifuged at8500 rpm for 20 minutes to clear the cell debris from the periplasmicextract. NANOBODIES were purified via cation exchange (Source 30Scolumn, wash buffer: 10 mM citric acid pH 4.0; Elution buffer 10 mMcitric acid/1M NaCl pH 4.0) followed by size exclusion chromatography(Superdex 75 Hiload 16/60 column; in d-PBS). The OD 280 nm was measuredand the concentration calculated. Samples were stored at −20 C.

RANKL13hum5 and RANKL13hum5_D62E were analysed for their bindingcapacity and neutralizing activity in Alphascreen as described inExample 2. Humanization of RANK13 and D62E mutation in RANKL13hum5 didnot interfere with potency of the NANOBODY. RANKL13, RANKL13hum5 andRANKL13hum5_D62E displayed similar potencies as measured in AlphaScreenassay (FIG. 7).

Example 10: Determination of Size of Linker Lengths in BivalentMolecules for Optimal Potencies RANKL13

A series of bivalent molecules containing different linker lengths wereconstructed. RANKL131 (RANKL13-9GS-RANKL13; SEQ ID NO: 643) and RANKL133(RANKL13-30GS-RANKL13; SEQ ID NO: 766) contain a 9GS or a 30GS linker,respectively. Both molecules were tested in AlphaScreen assay and FMATassay and compared to the monovalent RANKL13 and the trivalentbispecific RANKL130. Calculated IC50 values are shown in Table C-14.Both assays indicate that a 9GS linker is sufficient to obtain a similarpotency as determined for RANKL130.

RANKL18

A series of bivalent molecules containing different linker lengths wereconstructed:

RANKL181biv: RANKL18-9GS-RANKL18 (SEQ ID NO: 657) RANKL182biv:RANKL18-20GS-RANKL18 (SEQ ID NO: 693) RANKL183biv: RANKL18-30GS-RANKL18(SEQ ID NO: 767) RANKL18hum6 Bi_25: RANKL18Hum6-25GS-RANKL18Hum6 (SEQ IDNO: 768)

RANKL 18hum6 Bi_30: RANKL18Hum6-30GS-RANKL18Hum6 (SEQ ID NO: 769)

All molecules were tested in AlphaScreen assay and FMAT assay andcompared to the monovalent RANKL18 and the trivalent bispecificRANKL180. Calculated IC50 values are shown in Table C-14. Both assaysindicate that a 30GS linker is required to obtain a similar potency asdetermined for RANKL180.

RANKL9

A series of bivalent molecules containing different linker lengths wereconstructed:

RANKL91biv: RANKL9-9GS-RANKL9 (SEQ ID NO: 636) RANKL92biv:RANKL9-20GS-RANKL9 (SEQ ID NO: 672) RANKL93biv: RANKL9-30GS-RANKL9 (SEQID NO: 770) RANKL94biv: RANKL9-15GS-RANKL9 (SEQ ID NO: 771)

All molecules were tested in AlphaScreen assay and FMAT assay andcompared to the monovalent RANKL9 and the trivalent bispecific RANKL90(SEQ ID NO: 708) (Table C-14). Both assays indicate that a 15GS linkeris sufficient to induce a shift in potency comparable to RANKL90.

Example 11: Construction and Production of Different Formats of theHumanized NANOBODIES

Bivalent and trivalent bispecific anti-RANKL NANOBODIES were constructedfrom RANKL13hum5 and RANKL18hum6. An overview of the different formattedhumanized NANOBODIES with corresponding NANOBODY IDs is represented inTable C-15.

11.1 Trivalent Bispecific NANOBODIES

The trivalent bispecific molecules (RANKL008a and RANKL010a) have twobuilding blocks corresponding to humanized anti-RANKL NANOBODIES with inthe middle a third humanized NANOBODY building block corresponding to ananti-Human Serum Albumin NANOBODY building block (ALB-12; SEQ ID NO:791). The individual building blocks are fused by a Gly/Ser (GGGGSGGGS;SEQ ID NO: 792) linker.

RANKL008a was expressed in E. coli and purified from medium andperiplasmic extracts. NANOBODY was captured on MabSelect Xtra (GEHealthcare, Uppsala, Sweden). Elution occurred with buffer-B (100 mMGlycine pH2.5). Fractions were neutralized with 1.5M Tris pH 7.5 anddialysed against 1/10 PBS. Samples were subsequently subjected to CationExchange using a Source 15 S column (GE Healthcare, Uppsala, Sweden).

RANKL010a was cloned into pPICZalphaA (Invitrogen, Carlsbad, Calif.) andtransformed in Pichia pastoris. Colonies were diluted in 250 ml BCGMmedium and grown at 30° C. At OD600 of 20-25, cultures were centrifugedand pellet resuspended in 80 ml of BMCM medium. Expression was inducedby addition of 100% methanol. NANOBODY was captured from the medium bycapturing on MabSelect Xtra (GE Healthcare, Uppsala, Sweden). Elutionfractions in 100 mM Glycine pH2.5 buffer were neutralised with 1.5M TrispH 7.5 and dialysed against 1/10 PBS. Sample was further purified byCation Exchange (Source 30 S column). Finally, a sizing step occurred onSuperdex 75 26/60 column.

11.2 PEGylated NANOBODIES

Bivalent RANKL13hum5 and RANKL18hum6 were constructed. Constructs wereexpressed in E. coli. Purification and pegylation was carried out asfollows: Bivalent NANOBODY was purified via Protein A affinitychromatography (MabSelect Xtra™). After elution, using 100 mM Glycine pH2.5, the collected sample was immediately neutralized using 1.5M TrispH7.5. After a Cation Exchange step (Source 15S column) the fractioncontaining the NANOBODY® was concentrated via Vivaspin (5 kD), DTT wasadded to a final concentration of 10 mM and incubated over night. Afterfree DTT was removed by SEC, PEG (3-Maleimidoproprionamide, 1,3-bis(Methoxy poly (ethylene glycol) modified 2-glycerol), Average MW 40,000;Nektar Therapeutics, San Carlos, Calif.) was added in a 5 molar excessand incubated overnight. The PEGylated NANOBODY was separated fromunPEGylated NANOBODY/free PEG via MacroCap SP cation exchange (buffer:25 mM Na-acetate pH5+0.5% pluronic). The bound proteins were eluted witha linear gradient to buffer (lx PBS+0.5% pluronic).

11.3 HSA Fusions

HSA fusion proteins, RANKL004h and RANKL006h, correspond to bivalentRANKL13hum5-9GS-RANKL13hum5 and RANKL18hum6-30GS-RANKL18hum6,respectively, which are C-terminally fused to HSA.

For the generation of RANKL004h, RANKL13hum5 was amplified by PCR usingfollowing primers:

SEQ ID Sequence NO GAAGTAGGATGGACGATGACAAACCCGCGAAGACTTTTCTGGTG 807GCGGGAGCGAGGTGCAGCTGGTGGA GTTCTATCGGGAAGACTTAGAACCTCCGCCGCCTGAGGAGACGG808 TGACCAG GGGTATCTCTCGAGAAAAGAGAGGTGCAGCTGGTGGAGTCTGGG 809TCTCTTCTCCTAGGTCTTTGAATCTGTGGGCGACTTCAGATTTATG 810AGCATCTGAGGAGACGGTGACCAG

Subsequent amplicons were digested using appropriate restriction enzymes(N-terminal fragment: BbsI/XhoI and C-terminal fragment: BbsI/Avr II)and were cloned into a XhoI/AvrII opened pPICZalphaA-HSA vector. Thisvector contains the coding sequence for full length human serum albumin.

For the generation of RANKL006h, RANKL18hum6 was amplified by PCR usingfollowing primers:

Sequence SEQ ID NO GGGTATCTCTCGAGAAAAGAGAGGTTCAGCTAGTGGAATCA 811CACCTCCGGATCCTCCACCTCCGCTACCTCCACCTCCACTG 812CCACCTCCACCTGAGGAGACGGTGACCAG GGTGGAGGATCCGGAGGTGGAGGTAGCGGAGGTGGAGGCTC813 AGGAGGTGGAGGCAGTGAGGTTCAGCTAGTGGAATCTCTTCTCCTAGGTCTTTGAATCTGTGGGCGACTTCAGAT 814TTATGAGCATCTGAGGAGACGGTGACCAG

Amplicons were digested using appropriate restriction enzymes(N-terminal fragment: BamHI/XhoI and C-terminal fragment: BamHI/Avr II)and were cloned into a XhoI/AvrII opened pPICZalphaA-HSA vector. Thisvector contains full length human serum albumin.

Plasmids were transformed to Pichia pastoris. Colonies were diluted in250 ml BCGM medium and grown at 30° C. Expression was induced byaddition of 100% methanol. NANOBODIES were purified from the medium bycapturing the NANOBODIES on MabSelect Xtra (GE Healthcare, Uppsala,Sweden) and further polishing steps using Poros50HQ and Superdex200XK26/60.

Example 12: Characterization of the Formatted Humanized NANOBODIES inAlphaScreen and FMAT Assay 12.1 Potency of the RANKL13hum5 Based Formatsin AlphaScreen and FMAT Assay

Trivalent bispecific NANOBODY RANKL008a, pegylated NANOBODY RANKL001pand HSA fusion protein RANKL004h were tested in AlphaScreen assay andFMAT assay and compared to the monovalent RANKL13hum5 and the wild typetrivalent bispecific RANKL130. Calculated IC50 values are shown in TableC-16. In both assays RANKL008a, RANKL001p and RANKL004h displayed asimilar potency that is comparable to that of RANKL130.

12.2 Potency of the RANKL18hum6 Based Formats in AlphaScreen and FMATAssay

Trivalent bispecific NANOBODY RANKL010a and pegylated NANOBODY RANKL003pwere tested in AlphaScreen assay and FMAT assay and compared to themonovalent RANKL18hum6 and the wild type trivalent bispecific RANKL180.Calculated IC50 values are shown in Table C-16. In both assays RANKL010aand RANKL003p displayed potencies that are comparable to that ofRANKL180.

12.3 Effect of Albumin on Binding Kinetics of RANKL13 with Anti-HumanSerum Albumin NANOBODY Building Block

An experiment was performed with wild type RANKL130NT(RANKL13-ALB-1-RANKL13) and humanized RANKL008a(RANKL13hum5-ALB-12-RANKL13hum5). In this experiment a 100 nM solutionwas passed over a chip with immobilized RANK-L. As shown in FIG. 8, thesensorgrams in the absence of albumin are comparable for both molecules(wild type and humanized) suggesting that both molecules interact in asimilar way with immobilized RANKL. Subsequently a mixture was prepared(100 nM of the NANOBODY with 500 nM HSA) and passed over the chip. Asthe signal was significantly higher for the complex, we conclude thatboth the wild type and humanized variants with anti-Human Serum AlbuminNANOBODY building block are able to bind to RANK-L and albuminsimultaneously.

12.4 Epitope Mapping of RANKL13hum5 and RANKL18hum6

In the past, experimental data were generated which supported thestatement that RANKL13hum5 and RANKL18hum6 bind to overlapping epitopes.This observation was confirmed in a BIACORE experiment. In these epitopemapping experiments the RANKL sensor chip was first saturated with thefirst NANOBODY (concentration of 500 nM). After 120 seconds,dissociation was allowed or a mixture containing the same concentrationof the first NANOBODY together with 500 nM of the NANOBODY to be testedwas injected for 120 seconds. In FIG. 9 the sensorgrams for RANKL18hum6and in FIG. 10 the sensorgrams for RANKL13hum5 are shown. Only a slightincrease in signal was observed upon injection of the mixture aftersaturation of the surface first with either RANKL13hum5 or RANKL18hum,indicating that RANKL13hum5 and RANKL18hum6 bind to overlappingepitopes.

12.5 Inhibition of RANK-L Induced Differentiation of RAW264.7 Cells byFormatted Humanized NANOBODIES

RAW264.7 cells (ATCC) were maintained in DMEM containing 10% FBS. Cellswere resuspended in alphaMEM without phenol red containing 10% FBS, 0.1%sodium pyruvate, 1% non essential amino acid and seeded at 2000cells/well in a 96-well plate. A dilution series of the formattedhumanized NANOBODIES was added to the wells. Differentiation toosteoclast-like cells was induced by adding 7.5 ng/mL RANKL (Peprotech,Rocky Hill, N.J.). The total tartrate-resistant phosphatase activity wasmeasured in the supernatant after 4 days using paranitrophenylphosphateas substrate (FIG. 11).

12.6 Inhibition of RANK-L Interaction with RANK by Formatted HumanizedNANOBODIES

96-well microtiter plates were coated overnight at 4° C. with anti-FcNANOBODY PMP02 and blocked with Superblock T20 (PBS) blocking buffer.Different concentrations of formatted humanized NANOBODIES werepreincubated with 200 ng/mL RANK-Fc and 5 ng/mL RANK-L after which themixtures were transferred to the coated wells. Bound RANK-L was detectedfor 1 h at room temperature (RT) with a 1/100 dilution of HumanTRANCE/RANKL/TNFSF11 Biotinylated Affinity Purified polyclonal antibody(R&D systems, Minneapolis, Minn.) in PBS containing 10% Superblock T20(PBS) blocking buffer followed by a 30 min incubation with horseradishperoxidase labeled streptavidin (1/5000 in PBS containing 10% SuperblockT20 (PBS) blocking buffer). Visualisation was performed with3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide after whichthe coloring reaction was stopped with 1N HCl. The absorbance wasdetermined at 450 nm. The inhibition of the RANK-L interaction with RANKby the formatted humanized NANOBODIES is shown in FIG. 12.

12.7 Binding of RANKL008a to Human Serum Albumin

96-well microtiter plates were coated overnight at 4° C. with humanserum albumin (HSA, 20 μg/mL in PBS). Wells were aspirated and blockedwith Superblock T20 (PBS) blocking buffer. Wells were incubated for 1 hat RT with a dilution series of RANKL008a. Subsequently, bound RANKL008awas detected with a bivalent NANOBODY coupled to horseradish peroxidase.Visualisation was performed as described above (FIG. 13).

12.8 Bifunctional Binding of RANKL008a to HSA and RANK-L

Two different ELISA formats were applied to measure a simultaneousbinding of RANKL008a to HSA and RANK-L.

In a first format, 96-well microtiter plates were coated overnight at 4°C. with neutravidin (2 μg/mL, PBS) and blocked with Superblock T20 (PBS)blocking buffer. Wells were incubated with biotinylated RANK-L (0.5μg/mL in PBS) after which a dilution series of RANKL008a was applied.Bound RANKL008a was detected with horseradish peroxidase labeled albumin(Genetex, San Antonio, Tex.; 1/6000). Visualisation was performed asdescribed above (FIG. 14A).

In a second format, 96-well microtiter plates were coated overnight at4° C. with HSA (10 μg/mL, PBS) and blocked with Superblock T20 (PBS)blocking buffer. Subsequently, a dilution series of RANKL008a wasapplied. After 1 h incubation at RT, bound RANKL008a was detected withbiotinylated RANK-L (5 ng/mL), followed by an incubation withhorseradish peroxidase labeled streptavidin (1/2000). Visualisation wasperformed as described above (FIG. 14B).

Example 13: Pharmacokinetics of Formatted Humanized NANOBODIES in Balb/cMice after a Single Intravenous or Subcutaneous Administration

A bolus dose of 100 μg of each NANOBODY was administered to male Balb/cmice (8 to 12 weeks) (n=3), either intravenously into the tail orsubcutaneously. At a series of time points, blood samples werecollected.

Detection of the different NANOBODIES was performed as follows: Maxisorbmicrotiter plates (Nunc, Wiesbaden, Germany) were coated for 1 hr atroom temperature (RT) with 100 μl of a 5 μg/ml solution of Neutravidine(Pierce, Rockford, Ill.) in PBS buffer. After coating, the plates wereaspirated for 4 seconds and blocked (300 μl/well) for 30 min at RT withSuperblock T20 (Thermo Scientific Pierce Protein Research Products,Rockford, Ill.). Plates were washed three times with PBS containing0.05% TWEEN20. After blocking, biotinylated human RANKL (0.25 μg/ml, 100μl/well) was captured for 1 hr at RT (600 rpm). Dilution series of theNANOBODY standards (prepared in PBS/0.1% casein) were spiked into 100%pooled blank mouse serum (Harlan, Oxon, United Kingdom) and were thenfurther diluted 1/10 with PBS containing 0.1% casein (in separatenon-coated plate Nunc), resulting in a final sample matrix consisting of10% pooled mouse serum. Serum samples were treated in the same way. Allpre-dilutions were incubated for 30 minutes at RT (600 rpm) in thenon-coated plate. After the capturing step, the coated plates werewashed three times (PBS containing 0.05% TWEEN20), and an aliquot ofeach sample dilution (100 μl) was transferred to the coated plate andallowed to bind for 1 hr at RT (600 rpm). After sample incubation, theplates were washed three times (PBS containing 0.05% TWEEN20) andincubated for 1 hr at RT with 100 μl of an in-house polyclonal rabbitanti-NANOBODY antibody (1 μg/ml, in PBS/0.1% casein). The plates werethen washed three times (PBS containing 0.05% TWEEN20) and incubatedwith 100 μl of a 1/2000 dilution (in PBS with 0.1% casein) of goatanti-rabbit HRP (DakoCytomation, Glostrup, Denmark). After incubationfor 30 minutes at RT (600 rpm), plates were washed three times (PBScontaining 0.05% TWEEN20) and incubated for 10 minutes in the dark with100 μl of es(HS)TMB (1/3 dilution in HRP Buffer; SDT, Brussels,Belgium). After 10 minutes, the reaction was stopped with 100 μl HCl(1N). After 5 minutes, the absorbance of each well was measured at 450nm (Tecan Sunrise spectrophotometer; Mannedorf, Switzerland), andcorrected for background absorbance at 620 nm Concentration in eachserum sample was determined based on a sigmoidal standard curve withvariable slope.

After iv administration, the serum concentration-time profile of eachmouse was subjected to a two-compartmental pharmacokinetic analysisusing the pre-programmed Model number 7 within WinNonlin ProfessionalSoftware Version 5.1 (Pharsight Corporation, Mountain View Calif., USA).Calculated parameters for the individual NANOBODIES are shown in TableC-17.

After sc administration, the serum concentration-time profile of eachmouse was subjected to a one-compartmental pharmacokinetic analysis(with first order absorption) using the pre-programmed Model number 3(no lag time) or 4 (with lag time) within WinNonlin ProfessionalSoftware Version 5.1 (Pharsight Corporation, Mountain View Calif., USA).Calculated parameters for the individual NANOBODIES are shown in TableC-18.

Example 14: Pharmacokinetics and Pharmacodynamics of Formatted HumanizedNANOBODIES in Cynomolgus Monkeys

Female cynomolgus monkeys were assigned to 17 groups, each groupconsisting of three individuals aged approximately 24 months.

Animals were dosed as described in Table C-19. NANOBODIES RANKL008a,RANKL001p and RANKL003p were administered at different doses (3 mg/kg;0.3 mg/kg; 0.03 mg/kg). NANOBODY ALB-8 served as a negative controlSmall molecule Ibandronate was included as a positive control. Serumsamples were taken for determination of the NANOBODY levels, antibodyanalysis, and analysis of the bone turnover marker serum N-telopeptide(serum N-Tx).

14.1 Pharmacokinetics

Concentrations of RANKL008a were determined in plasma as follows:96-well microtiter plates (Maxisorp, Nunc, Wiesbaden, Germany) werecoated overnight at 4° C. with 100 μL neutravidin (2 μg/mL, Pierce,Rockford, Ill.). Wells were aspirated and blocked for 30 min at RT with300 μL SuperBlock®T20 PBS (Pierce, Rockford, Ill.). After 3 washingsteps with PBS-0.05% TWEEN20, biotinylated RANKL (0.5 μg/mL in PBS-0.1%casein-0.05% TWEEN20, in-house) was captured by incubating 100 μL for 1hr at RT while shaking at 600 rpm. After this incubation step, wellswere washed 3 times with PBS-0.05% TWEEN20. The standards, QC andpredilutions of the test samples were prepared in a non-coated(polypropylene) plate in 100% Cynomolgus monkey plasma and incubated for30 min at RT while shaking at 600 rpm. A 1/10 dilution of the samples inPBS-0.1% casein-0.05% TWEEN20 (final concentration of Cynomolgus monkeyplasma is 10%) was transferred to the coated plate and incubated for 1 hat RT while shaking at 600 rpm. After three washing steps with PBS-0.05%TWEEN20, the plates were incubated with the purified in-house rabbitanti-NANOBODY polyclonal antibody (1 μg/mL in PBS-0.1% casein-0.05%TWEEN20, in-house, Batch No. 15/05/06) for 1 hr at RT while shaking at600 rpm. After 3 washing steps with PBS-0.05% TWEEN20, 100 μl horseradish peroxidase (HRP) labeled polyclonal goat anti-rabbit (1/5000 inPBS-0.1% casein-0.05% TWEEN20, DakoCytomation, Glostrup, Denmark;Article No. P0448) was incubated for 1 h at RT while shaking at 600 rpm.Visualization was performed covered from light for 10 min with 100 μL3,3′,5,5′-tetramethylbenzidine (esTMB, SDT, Brussels, Belgium). Thissubstrate was 1/3 diluted in substrate buffer. The substrate buffer is acomposition of 60% Na₂HPO₂ (100 mM) and 40% citric acid (100 mM). After10 min, the colouring reaction was stopped with 100 μL 1N HCl. Theabsorbance was determined at 450 nm after a 10 sec shake in the TecanELISA reader, and corrected for background absorbance at 620 nmConcentration in each sample was determined based on a sigmoidalstandard curve.

Concentrations of RANKL001p and RANKL003p were determined as follows:96-well microtiter plates (Maxisorp, Nunc, Wiesbaden, Germany; ArticleNo. 430341) were coated overnight at 4° C. with 100 μL neutravidin (2μg/mL, Pierce, Rockford, Ill.). Wells were aspirated and blocked for 30min at RT with 300 μL SuperBlock®T20 PBS (Pierce, Rockford, Ill.). After3 washing steps with PBS-0.05% TWEEN20, biotinylated RANKL (0.5 μg/mL inPBS-0.1% casein-0.05% TWEEN20, in-house) was captured by incubating 100μL for 1 hr at RT while shaking at 600 rpm. After this incubation step,wells were washed 3 times with PBS-0.05% TWEEN20. The standards, QC andpredilutions of the test samples were prepared in a non-coated(polypropylene) plate in 100% Cynomolgus monkey plasma and incubated for30 min at RT while shaking at 600 rpm. A 1/10 dilution of the samples inPBS-0.1% casein-0.05% TWEEN20+10% Cynomolgus monkey plasma (finalconcentration of Cynomolgus monkey plasma was 10%) was transferred tothe coated plate and incubated for 1 h at RT while shaking at 600 rpm.After three washing steps with PBS-0.05% TWEEN20, the plates wereincubated with the purified in-house rabbit anti-NANOBODY polyclonalantibody (1 μg/mL in PBS-0.1% casein-0.05% TWEEN20, in-house) for 1 hrat RT while shaking at 600 rpm. After 3 washing steps with PBS-0.05%TWEEN20, 100 μl horse radish peroxidase (HRP) labeled polyclonal goatanti-rabbit (1/2000 in PBS-0.1% casein-0.05% TWEEN20, DakoCytomation,Glostrup, Denmark) was incubated for 1 h at RT while shaking at 600 rpm.Visualization was performed covered from light for 10 min with 100 μL3,3′,5,5′-tetramethylbenzidine (undiluted esTMB, SDT, Brussels,Belgium). After 10 min, the colouring reaction was stopped with 100 μL1N HCl. The absorbance was determined at 450 nm after a 10 sec shake inthe Tecan ELISA reader, and corrected for background absorbance at 620nm Concentration in each sample was determined based on a sigmoidalstandard curve.

Individual plasma concentration-time profiles of all monkeys weresubjected to a non-compartmental pharmacokinetic analysis (NCA) usingWinNonlin Professional Software Version 5.1 (Pharsight Corporation,Mountain View Calif., USA). The pre-programmed Model 200 or 201 was usedafter subcutaneous and intravenous dosing respectively. The AUC andderived PK-parameters were calculated by means of the linear-up/log downtrapezoidal rule. An overview of the calculated pharmacokineticparameters is presented in Tables C-20 to C-23.

14.2 Pharmacodynamics

Changes in bone resorption induced by the NANOBODIES were assessed byassaying serum NTx using immunoassays according to manufacturer'sinstructions (Osteomark® NTx serum, Wampole Laboratories).

FIGS. 15A-15 and 16A-16C represent the serum NTx concentration-timeplots for the different NANOBODIES dosed at different amounts eitherintravenously or subcutaneously.

All plasma concentration-time profiles of each NANOBODY were fittedsimultaneously to the pharmacokinetic function that was minimallynecessary to provide a reasonable characterization of theconcentration-time data. Only the plasma concentration-time profiles ofthe monkeys devoid of significant immunogenicity were included in theanalysis. A two-compartmental pharmacokinetic model with both linear andnon-linear clearance from the central compartment was found tocharacterize the dose and time dependent pharmacokinetics. In turn,these NANOBODY-specific pharmacokinetic functions were used as an inputfunction for the pharmacodynamic model to estimate the in vivo potency(IC₅₀) and intrinsic activity (I_(max)) on serum NTx turnover.

The intrinsic activity (I_(max)) and potency (IC₅₀) of each NANOBODY onthe serum NTx turnover was described using an indirect response model.The indirect response model was parameterized with the half-life ofserum NTx (t_(1/2)=0.693/k_(out)) and a NANOBODY-mediated inhibition(Hill function) on the zero order production rate of NTx (K_(in)). TheNANOBODIES were assumed to inhibit K_(in) by means of a Hill equationparameterized with I_(max), IC₅₀ and a shape factor n. For each NANOBODYa single set of PD parameters was estimated, except for RANKL003p wherethe turnover parameters (K_(in), K_(out)) were allowed to vary for eachdose level.

The obtained pharmacodynamic parameters of all NANOBODIES are presentedin Table C-24. Although all three NANOBODIES had similar intrinsicactivity and mediated an almost complete inhibition of serum NTx(I_(max)≈90), their potency was significantly different. RANKL008a wasfound to be the most potent inhibitor of serum NTx synthesis, followedby RANKL001p and RANKL003p. RANKL003p had a tenfold lower potency thanRANKL008a. The average serum NTx half-life was approximately 1.6 hr.

Example 15: Cross-Reactivity of RANKL 130, RANKL180, RANKL008a,RANKL001p and RANKL003p with TNF Family Members TNFα, CD40 Ligand(CD40L) and TRAIL

The cross-reactivity of the anti-RANK-L NANOBODIES with TNF familymembers TNFα, TRAIL or to mouse RANK-L was tested in a competitionELISA. Human RANK-L was coated on 96-well plates as described in Example2. NANOBODIES (100 pM) were preincubated with varying concentrations ofhuman RANK-L, mouse RANK-L, TNFα or TRAIL (10 nM down to 13.7 pM) beforethey were added to the plates. Binding of the NANOBODIES to the RANK-Lcoated plates was only inhibited by exogenously added RANK-L and was notaffected by the addition of the other ligands (FIG. 17).

Example 16. Administration of Anti-RANK-L NANOBODIES Prevents Bone Lossand Maintains Strength and Quality in Ovariectomized Cynomolgus Monkeys

Osteoporosis is characterized by low bone mass and microarchitecturaldeterioration of bone tissue, with a consequent increase in fragilityand susceptibility to fractures. Estrogen depletion contributes to thelow bone mass characteristic of postmenopausal osteoporosis. Therefore,estrogen depletion has been used as a bone loss animal model forstudying osteoporosis therapies

One month after OVX or sham surgery, ovariectomized (OVX) cynomolgusmonkeys (cynos) (9 to 16 years old) are treated with vehicle oranti-RANK-L NANOBODY for 16 months. Sham controls are treated withvehicle. Effects on bone resorption and on bone mineral density (BMD)upon administration of the anti-RANK-L NANOBODY are analysed.

After sacrifice, ex vivo DXA and pQCT scans are taken of the intactright femur, L3-L4 vertebral bodies, and L5-L6 vertebral 5 mm cancellouscores. Destructive testing is performed by 3-point bending of the femurdiaphysis and humeral cortical beams, shearing of the femur neck, andcompression of the lumbar vertebral specimens.

The effects of the anti-RANK-L NANOBODY on bone turnover at thehistologic level, and their relationships with bone strength areanalysed as follows: Double fluorochrome labels are injected prior toiliac and rib biopsies (at month 6 and 12), and prior to sacrifice.Histomorphometry is performed on these biopsies, the tibial diaphysis,and cancellous bone in L2 vertebra and the proximal femur.

Example 17. Construction and Analysis of VHH-Fc Fusions

NANOBODIES directed against RANK-L are cloned in a suitable vector togenerate genetic fusions to the CH1 deleted Fc portion of human IgG1 orof human IgG2. The hinge regions linking the NANOBODY to Fc are derivedeither from IgG1 or IgG2.

Plasmid constructs are transfected in eukaryotic cell lines and Fcfusion is secreted into the culture supernatant. Products are purifiedand analysed on a Coomassie stained gel. Representative sequences aredepicted in Table B-6 (SEQ ID NO's: 774-789). NANOBODY-fusions aretested in FMAT competition binding assay, NF-κB reporter assay and TRACP5b osteoclast differentiation assays.

Example 18. Cross-Reactivity of RANKL008a with TNF Family Members TNF-α,TRAIL, FasL, CD40L, LTα1/β2, LTα2/β1 and LIGHT

In order to identify human proteins that share sequence similarity withRANK-L, its protein sequence (Genbank accession number CAM23721) wasused to search the translated human genome with TBLASTN using thestandard settings (BLOSUM62). For the highest scoring sequences, theregion corresponding to the CD-loop was compared to RANK-L to assesspossible binding of RANKL008a.

Cross-reactivity of RANKL008a with selected TNF family members wastested via competition ELISA. Ninety six-well microtiter plates (Nunc,Maxisorp) were coated ON at 4° C. with 100 μL of 2 μg/mL neutravidin(Pierce) in PBS. Wells were aspirated and blocked with 300 μL ofSuperBlock T20 PBS blocking buffer (Pierce) for 30 min at RT. Wells werewashed thrice with 0.05% PBS-TWEEN, and subsequently incubated with 0.05μg/mL biotinylated sRANK-L diluted in PBS-TWEEN for 1 h at RT and 600RPM. In the mean time, 3.27 ng/mL of RANKL008a was preincubated with adilution series of TNF receptor family members (RANK-L, TNF-α, TRAIL,FasL, CD40L, LTα1/β2, LTα2/β1 and LIGHT) for 1 h at RT and 600 RPM. ThesRANK-L-containing wells were washed 3 times with PBS-TWEEN and 100 μLof the pre-incubated RANKL008a samples was transferred to the coatedplate. After 1 h incubation at RT and 600 RPM, bound RANKL008a wasdetected for 30 min at RT and 600 RPM with 100 μL of polyclonal rabbitanti-VHH R23 (1 μg/mL in PBS-TWEEN, Ablynx). The wells were washed asabove and incubated for 30 min at RT and 600 RPM with HRP-conjugatedpolyclonal goat anti-rabbit Ig (0.15 μg/mL in PBS-TWEEN,DakoCytomation). Visualisation was performed with esTMB for 7 min, afterwhich the reaction was stopped with 1N HCl. The absorbance wasdetermined at 450 nm.

Binding of the NANOBODIES to the plates was only inhibited byexogenously added unlabeled RANK-L and was not affected by the additionof the other ligands (FIGS. 18A-18C).

Example 19. RANKL-mediated differentiation of CD14+ monocytes PeripheralBlood Mononuclear cells (PBMCs) were purified from human buffy coatsusing Ficoll Paque. CD14+ monocytes were isolated using CD14 Microbeads(Miltenyi Biotec) according to the manufacturer's instructions. TheCD14+ cell fraction and the CD14-fraction (i.e. the effluent during theseparation procedure; cells not attached to the anti-CD14 beads) wereseeded at 100.000 cells/well in a 96-well culture plate in aMEM culturemedium without phenol red containing 10 vol % FBS, 0.1 vol %penicillin/streptomycin, 0.1 vol % sodium pyruvate and 1 vol % nonessential amino acids. After 2 hours incubation at 37° C. and 5% CO₂,NANOBODIES were added and differentiation was induced with 7.5 ng/mLrecombinant human RANKL and 50 ng/mL human Macrophage colony-stimulatingfactor (MCSF). Cells were cultured for 11 days and 50 vol % of theculture medium was replaced every 2 days by fresh medium.Differentiation was assessed by cytochemical staining for intracellularTRACP performed after fixation in 3.7% formaldehyde in acetone using theLeukocyte Acid Phosphatase kit (Sigma). Moreover, TRACP 5b isoformactivity in the culture medium was performed using the BONETRAP® assay(Immunodiagnostic Systems) according to the manufacturer's instructions.

RANKL008a completely blocked RANKL-mediated differentiation of CD14+monocytes to osteoclasts. Indeed, in the presence of effectiveconcentrations of RANKL008a, RANKL-induced intracellular TRACP stainingwas inhibited (FIGS. 19A-19C). Moreover, RANKL-induced TRACP5b activityin conditioned medium was completely blocked (FIG. 20). In the absenceof RANKL or MCSF, baseline TRACP5b activity was measured, as expected.

Example 20: Inhibition of Membrane RANKL (mRANKL) Interaction withRANK-Fc

Interaction of mRANKL with RANK was measured using a cell adhesionassay. For this purpose, Chinese Hamster Ovary (CHO) cells which areexpressing recombinant membrane-bound human RANKL were used. Cells weremaintained in RPMI containing Glutamax, 25 mM Hepes and 10 vol % FBS.

96-well white polystyrene plates (Corning) were immobilized with theanti-Fc NANOBODY PMP02 (5 μg/mL in sodium bicarbonate buffer pH 9.6).Wells were blocked with PBS containing 3% BSA for 1 h at RT and washedwith PBS. Cells were detached with non-enzymatic dissociation buffer andresuspended in RPMI1640 medium. After 30 min incubation at RT of cells(400.000/mL) with 500 ng/mL RANK-Fc in the presence or absence ofNANOBODIES or controls, the mixtures were transferred to the coatedwells. After 2 h incubation at RT, wells were washed once with PBS andadhered cells were visualized using the luminescent-based CellTiter Glo®reagent for cell viability (Promega) according to the manufacturer'sinstructions.

RANKL008a could concentration-dependently and completely block theinteraction of mRANKL with RANK-Fc (FIG. 21). A clear shift in potencywas observed for CabLys3-Alb11-13H5 (SEQ ID NO: 815) compared toRANKL008a, while 13H5-CabLys3-13H5 (SEQ ID NO: 816) displayed similaractivity.

Example 21: Inhibition of mRANKL Mediated Osteoclast Differentiation

Osteoclast differentiation mediated by mRANKL was investigated using acoculture assay in which RAW264.7 cells were stimulated with recombinantmembrane bound human RANKL expressed on CHO cells. RAW264.7 cells andCHO cells were maintained as described above. CHO cells were detachedusing non-enzymatic dissociation buffer and resuspended in RPMI1640containing 10 vol % FBS and 1 vol % penicillin/streptomycin. CHO cellswere seeded at a density of 20.000 cells/well in a 96-well microtiterplate and incubated for 24 h at 37° C. and 5% CO₂. Cells were washed 3times with PBS and subsequently fixed in a solution of 3.7%paraformaldehyde in PBS. A dilution series of NANOBODIES or controls wasadded to the wells. Subsequently, wells were incubated with RAW264.7cells for 5 days at 37° C., 5% CO₂ and at a density of 2000 cells/wellin aMEM culture medium without phenol red containing 10 vol % FBS, 0.1vol % penicillin/streptomycin, 0.1 vol % sodium pyruvate and 1 vol % nonessential amino acids. Differentiation was assessed by eithercytochemical staining for intracellular TRACP or TRACP activitymeasurements in the conditioned medium as described above (FIGS. 22 and23A-23C).

Example 22: Pharmacokinetics and Pharmacodynamics of RANKL008a Vs.αOPGL-1 in Cynomolgus Monkeys

RANKL008a was expressed in Pichia pastoris and purified on SP Sepharoseas a capturing step and a Q filter as a polishing step or on SPSepharose as a capturing step and Capto MMC as a polishing step (GEHealthcare Life Sciences). Concentration of the NANOBODY and bufferswitch to PBS was done via UF/DF (Sartorius Hydrosart Sartocon Slice200, 10 kDa). A final filtration was carried out at 0.22 μm.

The amino acid sequence of the monoclonal antibody αOPGL-1 is describedin SEQ ID NO's: 2 and 4 of US2004/0033535. αOPGL-1 was expressed inCHO-K1SV cells (Lonza) and purified on MabSelect Sure as a capturingstep and Source30S as a polishing step (GE Healthcare Life Sciences).

Female Cynomolgus monkeys (3 animals per test compound) received asingle i.v. bolus administration of RANKL008a at 0.3 mg/kg or αOPGL-1 at1 mg/kg. An additional three female monkeys were dosed with PBS andserved as negative control.

Blood samples were processed to plasma for determination of the NANOBODYlevels and antibody analysis. In addition, blood samples were collectedand processed to serum for the analysis of the bone turnover markerserum N-telopeptide (serum N-Tx).

22.1 Bioanalytical Determination of RANKL008a and αOPGL-1 22.1.1Determination of RANKL008a Plasma Concentrations

Plasma concentrations of RANKL008a were determined in plasma using aRANKL-independent ELISA. In short, 96-well microtiter plates (Maxisorp,Nunc, Article No. 430341) were coated overnight at 4° C. with 100 μl ofan in-house made anti-NANOBODY NANOBODY at a concentration of 3 μg/ml inbicarbonate buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH9.6). Wells wereaspirated and blocked for 30 min at room temperature (RT) with 300 μlSuperBlock®T20 (PBS) (Pierce, Article No. 37516, Lot#JG121073). Thestandard curve stock solution and the QC samples were prepared in a lowadhesion plate (Nunc Article No. 24994) in 100% pooled Cynomolgus plasma(Sera Laboratories International; SLI#FF109188/209188;catalog#PSCIMI-118-M; aliquoted on 3 Oct. 2008) and incubated for 1 h atRT while shaking at 600 rpm. A 1/20 dilution of the standard stocksolution, QCs and test samples were made in PBS/0.1% casein (resultingin a 5% final plasma concentration). Further dilutions of thecalibrators and test samples were made in PBS/0.1% casein supplementedwith 5% plasma. After the 1/20 dilution, test samples were furtherdiluted in order to bring them into the dynamic range. The 5% plasmasolutions were incubated at RT and 600 rpm for 30 minutes. 100 μl/wellwas transferred to the coated and blocked plate and incubated for 2 hrat RT while shaking at 600 rpm. The plates were washed 3 times withPBS/0.05% TWEEN20. 100 μl of an in-house made anti-NANOBODY NANOBODY at250 ng/ml in PBS/0.1% casein was added per well and plates wereincubated for 1 h at RT while shaking at 600 rpm. Plates were washed 3times with PBS/0.05% TWEEN20. 100 μl strep-HRP (1/10000 in PBS/0.1%casein, Dako: P0397; Batch no 00032671) was added per well and plateswere incubated for 30 min at RT while shaking at 600 rpm. Again, plateswere washed 3 times with PBS/0.05% TWEEN20 and 100 μl of the3,3′,5,5′-tetramethylbenzidine substrate (es(HS)TMB (SDT-reagents)) 1/3diluted in substrate buffer (HRP buffer: 60% 100 mM Na₂HPO₄.2H₂O, 40%citric acid, pH 4.3)) was added per well. The colouring reaction wasallowed to proceed for 15 minutes at RT in the dark, after which thereaction was stopped by adding 100 μl 1N HCl per well. The absorbancewas determined at 450 nm after a 10 seconds shake in the Tecan ELISAreader, and corrected for background absorbance at 620 nm. Theconcentration in each sample was determined based on a 4 parameterlogistic fit of the standard curve.

The assay was validated and assay range was verified to be between 25 to600 ng/ml on plasma level. All samples were analysed in duplicate and areported value was generated by calculating an average concentrationfrom the replicates from the lowest dilution factor. All assaysgenerating a reported value passed the 4/6/20 rule on freshly preparedQC samples (% bias and % CV of 4 out of 6 QC samples should be ≦20% withleast 50% of the QC samples passing this criterion at each concentrationlevel). If the 4/6/20 rule was not met or the % CV of the duplicatestudy samples was >20%, no reported value was generated and the sampleswere re-analyzed. Incurred sample re-analysis, performed on 10% of thestudy samples showed that reported values did not differ more than 30%upon re-analysis.

22.1.2 Determination of αOPGL-1 Plasma Concentrations

Plasma concentrations of αOPGL-1 were determined using ELISA asdescribed below.

96-well microtiter plates (Maxisorp, Nunc, Article No. 430341) werecoated overnight at 4° C. with 100 μl neutravidin (Thermo Scientific,Prod#31000, Lot#JC122585, stock conc: 2 mg/ml) at a concentration of 2μg/ml in PBS (Phosphate buffered saline). Wells were aspirated andblocked for 30 min at room temperature (RT) with 300 μl PBS/1% casein(casein: Calbiochem; catalog nr: 218682). Afterwards plates were washed3 times with PBS/0.05% TWEEN20 and 100 μl biotinylated RANKL (Peprotech,Cat#310-01; biotinylated in-house) was added per well. The plates wereincubated for 1 h at RT while shaking at 600 rpm. The standard curvestock solution and the QC samples were prepared in a low adhesion plate(Nunc Article No. 24994) in 100% pooled Cynomolgus plasma (SeraLaboratories International; SLI#FF109188/209188; catalog#PSCIM-118-M;aliquoted on 3 Oct. 2008) and incubated for 1 h at RT while shaking at600 rpm. A 1/20 dilution of the standard stock solution, QCs and testsamples was made in PBS/0.1% casein (resulting in a 5% final plasmaconcentration). Further dilutions of the calibrators and test sampleswere made in assay diluents (PBS/0.1% casein supplemented with 5%plasma). If needed, test samples were further diluted to bring them intothe dynamic range of the assay. The 5% plasma solutions were incubatedat RT and 600 rpm for 30 minutes. 100 μl/well was transferred to thebio-RANKL containing plate and incubated for 2 h at RT while shaking at600 rpm. The plates were washed 3 times with PBS/0.05% TWEEN20. 100 μlof rabbit anti-human IgG-HRP (Dako; P0214; Lot#012(101); 1/20000 inPBS/0.1% casein) was added per well and plates were incubated for 1 h atRT while shaking at 600 rpm. Plates were washed 3 times with PBS/0.05%TWEEN20 and 100 μl of the 3,3′,5,5′-tetramethylbenzidine substrate (ESTMB (SDT-reagents) 1/3 diluted in substrate buffer (60% 100 mMNa₂HPO₄.2H₂O, 40% citric acid, pH 4.3)) was added per well. Thecolouring reaction was allowed to proceed for 15 minutes at RT in thedark, after which the reaction was stopped by adding 100 μl 1N HCl perwell. The absorbance was determined at 450 nm after a 10 seconds shakein the Tecan ELISA reader, and corrected for background absorbance at620 nm. The concentration in each sample was determined based on a 4parameter logistic fit of the standard curve.

The assay was validated and the assay range was verified to be between60 to 800 ng/ml on plasma level. All samples were analysed in duplicateand a reported value was generated by calculating an averageconcentration from the replicates from the lowest dilution factor. Allassays generating a reported value passed the 4/6/25 rule on freshlyprepared QC samples (% bias of 4 out of 6 QC samples should be ≦25% withleast 50% of the QC samples passing this criterion at each concentrationlevel). If the 4/6/25 rule was not met or the % CV of the duplicatestudy samples was >20%, no reported value was generated and the sampleswere re-analyzed. Incurred sample re-analysis, performed on 10% of thestudy samples showed that reported values did not differ more than 30%upon re-analysis.

22.2 Determination of Anti-Drug Antibodies 22.2.1 Determination ofAnti-Drug Antibodies Against RANKL008a in the Plasma of the CynomolgusMonkeys

Cynomolgus monkey plasma was screened for anti-drug antibodies againstRANKL008a and its building blocks as described below.

Selected monkey plasma samples at predose and at various time pointsafter RANKL008a administration were screened for the presence ofanti-drug antibodies using an ELISA-based direct detection titrationassay. 96-well microtiter plates (Maxisorp, Nunc, Article No. 430341)were coated overnight at 4° C. with either RANKL008a (Pichia produced asdescribed above; 1.42 mg/ml), the RANKL13H5 building block or the Alb8building block, each at a concentration of 5 μg/mL in bicarbonate buffer(15 mM Na₂CO₃ (1.59 g/l)+35 mM NaHCO₃ (2.93 g/l) in 1 l, pH 9.6) (100 μLper well). Wells were aspirated and blocked for 30 minutes at roomtemperature (RT) with 300 μL SuperBlock®T20(PBS) (Pierce, Article No.37516, Lot#JG121073). Afterwards plates were washed 3 times withPBS/0.05% TWEEN20. Plasma test samples and positive controls (plasmacollected from Cynomolgus monkey with positive response to NANOBODY)were prepared in a low adhesion plate (Nunc Article No. 249944) by afirst 1/50 dilution step, followed by a 1/3 dilution series in 6 stepsin PBS/0.1% casein/0.05% TWEEN20. 100 μl/well samples and controls weretransferred to the coated plate and were allowed to bind for 1 h at RTwhile shaking at 600 rpm. Negative control for each plate was PBS/0.1%casein/0.05% TWEEN20 (buffer). The plates were washed 3 times withPBS/0.05% TWEEN20. 100 μL of Rabbit anti-Monkey IgG-Bio (1/5000 inPBS/0.1% casein/0.05% TWEEN20; Nordic Immunology; RAMon/IgG(H+L)/Bio(5086)) was added per well and plates were incubated for 30 min at RTwhile shaking at 600 rpm. Afterwards plates were washed 3 times withPBS/0.05% TWEEN20 and 100 μl streptavidin-HRP (1/5000 in PBS/0.1%casein/0.05% TWEEN20; Dako; P0397; Lotnr. 00047645) was added per well.Plates were incubated for 30 min at RT while shaking at 600 rpm. Again,plates were washed 3 times with PBS/0.05% TWEEN20 and 100 μl of the3,3′,5,5′-tetramethylbenzidine substrate (ES TMB (SDT-reagents) 1/3diluted in substrate buffer (60% 100 mM Na₂HPO₄.2H₂O, 40% citric acid,pH 4.3)) was added per well. The colouring reaction was allowed toproceed for 15 minutes at RT in the dark, after which the reaction wasstopped by adding 100 μl 1N HCl per well. The absorbance was determinedat 450 nm after a 10 seconds shake in the Tecan ELISA reader, andcorrected for background absorbance at 620 nm.

The titer was determined as the highest dilution factor at which the ODlevel was above threshold. The threshold was defined as the mean of thenegative controls multiplied by 2.

22.2.2. Determination of Anti-Drug Antibodies Against αOPGL-1 in thePlasma of the Cynomolgus Monkey

Cynomolgus monkey plasma was screened for anti-drug antibodies againstαOPGL-1 as described below.

Selected monkey plasma samples at predose and at various time pointsafter αOPGL-1 administration were screened for the presence of anti-drugantibodies (IgG isotype) using an ELISA-based bridging assay. 96-wellmicrotiter plates (Maxisorp, Nunc, Article No. 430341) were coatedovernight at 4° C. with αOPGL-1 (at a concentration of 5 μg/mL in PBS(Phosphate-buffered saline) buffer (10 PBS tablets (Oxoid/BR0014G) perlitre MQ) (100 μL per well). Wells were aspirated and blocked for 30 minat room temperature (RT) with 300 μL SuperBlock®T20(PBS) (Pierce,Article No. 37516, Lot#JG121073). Afterwards plates were washed 3 timeswith PBS/0.05% TWEEN20. Plasma test samples were prepared in a lowadhesion plate (Nunc Article No. 249944) by a 1/2 dilution step inPBS/0.1% casein. 100 μl/well samples were transferred to the coatedplate and allowed to bind for 1 h at RT while shaking at 600 rpm.Negative control for each plate was PBS/0.1% casein (buffer). The plateswere washed 3 times with PBS/0.05% TWEEN20. 100 μL of biotinylatedαOPGL-1 at a concentration of 1 μg/mL in PBS/0.1% casein was added perwell and plates were incubated for 30 min at RT while shaking at 600rpm. Afterwards plates were washed 3 times with PBS/0.05% TWEEN20 and100 μl streptavidin-HRP (1/5000 in PBS/0.1% casein; Dako; P0397; Lotnr.00047645) was added per well and plates were incubated for 30 min at RTwhile shaking at 600 rpm. Again, plates were washed 3 times withPBS/0.05% TWEEN20 and 100 μl of the 3,3′,5,5′-tetramethylbenzidinesubstrate (ES TMB (SDT-reagents) 1/3 diluted in substrate buffer (60%100 mM Na₂HPO₄.2H₂O, 40% citric acid, pH 4.3)) was added per well. Thecolouring reaction was allowed to proceed for 15 minutes at RT in thedark, after which the reaction was stopped by adding 100 μl 1N HCl perwell. The absorbance was determined at 450 nm after a 10 seconds shakein the Tecan ELISA reader, and corrected for background absorbance at620 nm.

22.3 Determination of Serum NTX Levels

For determination of the serum NTx levels in the serum of the Cynomolgusmonkeys the Osteomark® NTx Serum kit (Wampole Laboratories, Princeton,N.J., USA) was used for the quantitative measure of cross-linkedN-telopeptides of type I collagen (NTx) in serum, being an indicator forbone resorption.

This Osteomark® NTx Serum kit is a competitive-inhibition enzyme-linkedimmunosorbent assay (ELISA) in which a synthetic NTx epitope is adsorbedonto a 96-well microplate. Diluted samples are added to the microplatewells, followed by a horseradish peroxidase labeled monoclonal antibody.NTx in the sample competes with the NTx epitope coated to the wells ofthe microplate. Following a wash step, the amount of bound labeledantibody is measured by the colorimetric generation of a peroxidesubstrate. Absorbance is determined spectrophotometrically and NTxconcentration calculated using a standard calibration curve withpurified NTx antigen. Assay values are reported in nanomoles BoneCollagen Equivalents per liter (nM BCE).

22.4. Data Analysis 22.4.1 Pharmacokinetic Data Analysis

Descriptive statistics (mean and SD) were calculated per dose group andper sampling time point using Microsoft Excel 2007. In case all threevalues were below the quantification limit (BQL), BQL was reported. Whenone or two out of three values were BQL, BQL values were set to zero andthe mean calculated.

Individual plasma concentration-time profiles were subjected tonon-compartmental analysis (NCA) (Model 201; i.v. bolus injection) usingWinNonlin Pro 5.1 (Pharsight Corporation, USA; 2006). The area under thecurve (AUC) was estimated using the lin up/log down rule. LLOQ valueswere treated as missing, except when comprised between two values abovethe LLOQ, then they were set to zero. The concentration at time zero(CO) was estimated through back-calculation based on the two first datapoints. The terminal elimination half-life (t½) was calculatedautomatically (best-fit) using a log-linear regression of the non-zeroconcentration-time data of the log-linear portion of the terminal phase.A minimum of three points were considered for the determination of λz.

The following main pharmacokinetic parameters were estimated: plasmaconcentration at time zero (C0); the area under the plasmaconcentration-time curve extrapolated to infinity (AUCinf), total bodyclearance (CL), volume of distribution at steady-state (Vdss), and thedominant half-life (t_(1/2)).

22.4.2 Pharmacodynamic Data Analysis

Descriptive statistics (mean and SD) were calculated per dose group andper sampling time point using Microsoft Excel 2007.

Response parameters and associated statistics for the overall timecourse were calculated by noncompartmental analysis of the response-timedata using WinNonlin Pro 5.1 (Pharsight Corporation, USA; 2006). Thenon-compartmental analysis was based on a model for pharmacodynamic data(Model 220). As threshold value 30% readout was considered. Thefollowing main pharmacodynamic parameters were determined: time belowthe threshold (Time below T), area under the threshold (AUC belowthreshold), time at which the serum NTx first drops below the threshold(tonset), and time at which the serum NTx first returns back above thethreshold (toffset).

22.5 Results 22.5.1 Immunogenicity Evaluation

Immunogenicity was observed in all animals. However, in combination withPK and PD data, only one animal from the αOPGL-1 treated-animalsdisplayed evident signs of immunogenicity, which likely have impactedthe PK/PD data. This animal was therefore not considered in subsequentPK and PD analyses.

22.5.2 Pharmacokinetics

In FIGS. 24A-24B, the mean plasma concentration time profiles ofRANKL008a and αOPGL-1 following an single i.v. bolus administration at0.3 mg/kg (RANKL008a) or 1 mg/kg (αOPGL-1), respectively in the femaleCynomolgus monkey are presented.

After i.v. injection of RANKL008a, the plasma levels of RANKL008adropped fairly rapidly up to about 1 day post-dose, corresponding to thedistribution phase of the compound. Thereafter the drug levels droppedgradually with two distinct elimination phases. The dominant half-lifewas ca 8 days.

After i.v. injection of αOPGL-1, plasma concentrations of αOPGL-1 alsodropped quite rapidly up to about 1 day post-dose, related to thedistribution phase of the compound. After the distribution phase,αOPGL-1 levels dropped more gradually with two distinct eliminationphases. The half-life of the dominant phase was ca 6 days.

In Table C-25 the main pharmacokinetic parameters of RANKL008a andαOPGL-1 following an single i.v. bolus administration with RANKL008a at0.3 mg/kg or αOPGL-1 at 1 mg/kg in the female Cynomolgus monkey arelisted.

22.5.3 Pharmacodynamics

In FIG. 25, the temporal time profiles of serum NTx concentrationsfollowing a single i.v. bolus administration at 0.3 mg/kg (RANKL008a) or1 mg/kg (αOPGL-1) in the female Cynomolgus monkey are shown.

The main pharmacodynamic parameters of RANKL008a and αOPGL-1 following asingle i.v. bolus dose of RANKL008a at 0.3 mg/kg or αOPGL-1 at 1 mg/kgin the female Cynomolgus monkey are listed in Table C-26,

Pharmacodynamic serum NTx sample analysis demonstrated that animalstreated with the RANKL008a (0.3 mg/kg) or αOPGL-1 (1 mg/kg) displayed arapid drop in serum NTx levels (mean T onset of 0.143 and 0.125 d,respectively in Table C-26). The maximum effect was observed at 8 dpost-dose for RANKL008a and somewhat later (11 d post-dose) afterαOPGL-1 administration. (Tmin values in Table C-26). After its maximumeffect serum Ntx levels slowly returned to baseline. The time below thethreshold (Time below T) was fairly comparable (RANKL008a: 19 d;αOPGL-1: 17 d) after dosing with RANKL008a (0.3 mg/kg IV) and αOPGL-1 (1mg/kg IV). Animals administered with PBS also showed a small buttransient drop in serum NTx levels. This drop was however never belowthe threshold value (30% of the baseline value) and was rather procedurerelated than a true test-item related effect.

TABLES

TABLE B-1 Preferred NANOBODIES against RANK-L (inhibitors of RANK/RANK-Linteraction) <Name, SEQ ID #; PRT (protein); ->Sequence >RANKL1|051_RANKL1cl25|9B1, SEQ ID NO: 560; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSGRTFSSSTMAWFRQPPGGERDFVASISTSGTRTLYADSVKGRFTISRDNAKSTGYLQMNSLKPEDTAVYFCAAVNRRGWEFWRLASGYDYWGLGAQVTVSS >RANKL2|051_RANKL2cl5|2B9,SEQ ID NO: 561; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKGLEWVSSIYSDGSTTDYADSVKGRFTISRDNAKNTLNLQMNSLKSEDTAVYYCAKDANSGGLEYDYWGQGTQVTVSS >RANKL3|051_RANKL3cl1|2G8,SEQ ID NO: 562; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL4|051_RANKL4cl23|4F12,SEQ ID NO: 563; PRT; ->KVQLVESGGGLVQTGDSLRLSCAASGRAIGSYAMGWFRQAPGKEREFVAVINYRGSSLKYADRVKGRFTISRDNAKNMVYLQMNSLKPDDTAVYYCAAQTSGADFGTTPQRYTYWGQGTQVTVSS >RANKL5|051_RANKL5cl10|4G8,SEQ ID NO: 564; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTIGGHTMAWFRQAPGKERDFVATITSSGSTIFYADSVKGRFTISRDNGKKTMTLEMDSLKPEDTAVYYCAARIRGKVTVDNFDYAYWGQGTQVTVSS >RANKL6|051_RANKL6cl8|4H9,SEQ ID NO: 565; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL7|051_RANKL7cl13|7B4,SEQ ID NO: 566; PRT; ->EVQLVESEGGPVQSGGSLRLSCAASGRTFSVSTIAWFRQAPGEGREFVAAIYPSGRNAYVADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAAYYCAAHQPSGSYYSAEAYAYWGQGTQVTVSS >RANKL8|051_RANKL8cl28|7E,SEQ ID NO: 567; PRT; ->EVQLVESGGGSVQPGGSLRLSCAASGGTFSRYAMGWFRQAPGKEREFVSAISVGGTYQYYVDSVKGRFTISRDNAESTVYLQMNSLKPEDTAVYYCAGDASPYGYLREYTATRFDYWGQGTQVTVSS >RANKL9|051_RANKL9cl32|8A11,SEQ ID NO: 568; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL10|RANKLPMP9B3|9B3,SEQ ID NO: 569; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGITFSSRTMGWFRQAPGKEREFVAAITPSSRTTYYADSVKGRFTISRDNAKNTVLLQMNSLKPEDTAVYYCAAERTYGSNYTRPTAWNYWGQGTQVTVSS >RANKL11|051_RANKL11cl16|9F10,SEQ ID NO: 570; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSKTMGWFRQPPGNEREFVAAITPTSRTTYYADSVKGRFTISRDNAKNTVSLQMNSLKFEDTAAYYCVAVRRYGSPPHDGSSYEYWGQGTQVTVSS >RANKL12|051_RANKL12cl1|9B6,SEQ ID NO: 571; PRT; ->EVQLVESGGGWMQAGGSLRLSCAASGRTFTMAWFRQAPGKEREFVAAITGSGRSTYYTDSVKGRFTISRDNAKNTAYLQMKSLKPEDTAVYYCAGLRGLGLEYDSAKSYSYWGQGTQVTVSS >RANKL13|051_RANKL13cl1|1C7,SEQ ID NO: 572; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL14|051_RANKL14cl1|6B8,SEQ ID NO: 573; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTSSYYTMSWFRQDPGKEREFVAAVPLSGNTYYADPVRGRFTISRDNAKNTADLQMNSLKPEDTAVYYCAARASGSIYNRGSYAYWGQGTQVTVSS >RANKL15|051_RANKL15cl1|7C5,SEQ ID NO: 574; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL16|051_RANKL16cl1|7G8,SEQ ID NO: 575; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASRRTFSSYAMGWFRQVPGKERDFVAAISTGSITIYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGKREPYLRQYTASNPYDYWGQGTQVTVSS >RANKL17|051_RANKL17cl1|9C2,SEQ ID NO: 576; PRT; ->EVQLVESGGGLVQVGDSLRLSCEASGRSRFSTYVMGWFRQAPGKEREFVAAVSWSSGNAYYIDSAKGRFATSRDTAKNIMYLQMNSLKPEDTAVYTCAAGRGYGLLSEYTQAPRYDYWGQGTQVTVSS >RANKL18|051_RANKL18cl1|7F11,SEQ ID NO: 577; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSS >RANKL19|051_RANKL19cl1|6C8,SEQ ID NO: 578; PRT; ->EVQLVESGGGLVQAGDSLRLSCAASGRTVTMGWFRQAPGKEREFVASITGSGSVTNYADSVKGRFTISRDNAKNTVFLQMNSLKPEDTAVYYCAAYLPSPYYSSYYDSTKYEYWGQGTQVTVSS >RANKL20|051_RANKL20cl1|2F4,SEQ ID NO: 579; PRT; ->EVQLVESGGGLVQAGDSLRLSCAASGRTFTMGWFRQAPGTEREFVAAISGSGKITNYADSVKGRFTISRDHAKNTVFLQMDSLKPEDTAVYYCAGYLRSPYYSSFYDSAKYEYWGQGTQVTVSS >RANKL21|051_RANKL21cl1|7C6,SEQ ID NO: 580; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASRRTFNSYAVGWFRQVPGEERDFVAAISTGSVTIYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGNREPYLRQYTASNPYDYWGQGTQVTVSS >RANKL22|051_RANKL22cl2|7D12,SEQ ID NO: 581; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASERTSRNYGMGWFRQAPGKEREFVAAITSAGGTTYYGDFVKGRFTISRDSAKYTVYLQMNSLKPEDTAVYWCAAKLQIGGRWHNLNDYGYRGQGTQVTVSS >RANKL23|051_RANKL23cl1|9H5,SEQ ID NO: 582; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGLTTVYTMAWFRQAPGKEREFVAAITRSGKTTYYADSVKGRFTISRDNAKNTVNLQMNSLKPDDTAVYYCAAKALLGMTNPAGYEYWGQETQVTVSS >RANKLPMP4B3, SEQ IDNO: 584; PRT; ->evqlvesgggwmqaggslrlscaasgrtftMAwfrqasgkerefvaAITGSGRSTYYTDSVKGrftisrdnakntaylqmkslkpedtavyycagLRGLGLEYDSAKSYSYwgqgtqvtvss >RANKLPMP2E11, SEQ IDNO: 585; PRT; ->EVXLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKLPMP2A6, SEQID NO: 586; PRT; ->evqlvesggglvqaggslrlscaasgltssRYTMSwfrqdpgkerefvaAVPLSGNTYYADPVRGrftisrdnakntvdlqmnslkpedtavyycaaRASGSIFNRGSYAYwgqgtqvtvss >RANKLPMP1F2, SEQ IDNO: 587; PRT; ->evqlvesggglvpaggslrlscaasgltdrRYTMSwfrqdpgkerefvaAVPLSGNTYYADPVRGrftisrdnakntvdlqmnslkpedtavyycaaRASGSIFNRGSYAYwgqgtqvtvss >RANKLPMP2D4, SEQ IDNO: 588; PRT; ->evqlvesggglvpaggslrlscaasgltdrRYTMSwfrqdpgkerefvaAVPLSGNTYYADPVRGrftisrdnakntvdlqmnslkpedtavyycaaRASGSIFNRGSYAYwgqgtqvtvss >RANKLPMP7B2, SEQ IDNO: 589; PRT; ->evqlvesggglvqaggslrlscaaaggtfrNYVMGwfrqapgkerefvtAISTGGSWTGYVDSVKDrftisrdntkntvylqmaslkpedtavyycaaTMPATTYLPRSERQYDYwgqgtqvtvss >RANKLPMP7A11,SEQ ID NO: 590; PRT; ->evqlvesggglvqaggsltlscaaagftfrRYVMGwfrqapgkerefvaAISTGGTWTGYVDSVKDrftisrdntkntvylqmaslkpedtavyncaaTTPTTSYLPRSERQYEYwgqgtqvtvss >RANKLPMP7F1, SEQID NO: 591; PRT; ->evqlvesggglvqaggslrlscaaagctfrNYVMGwfrqapgkerefvtAISTGGTWTGYVDSVKDrftisrdntkntvnlqmaslkpedtavyycaaTTPTTSYLPRSERQYEYwgqgtqvtvss >RANKLPMP7H5, SEQID NO: 592; PRT; ->evqlvesggglvqaggslrlscaaaggtfrNYVMGwfrqapgkerefvaAISTGGSWTGYVDSVKDrftisrdntkntvylqmvslkpedtavyycaaTTPATTYLPRSERQYDYwgqgtqvtvss >RANKLPMP7E7, SEQID NO: 593; PRT; ->evqlvesggglvqaggslrlscaaaggtfrNYVMGwfrqapgkerefvtAISAGGSWTGYVDSVKDrftisrdntkntvylqmaslkpedtavyycaaTTPATTYLPRSERQYDYwgqgtqvtvss >RANKLPMP7E2, SEQID NO: 594; PRT; ->evqlvesggglvqaggslrlscaaagytfrAYVMGwfrqapgkerefvaGISTGGTWTGYVDSVKDrftisrdntkntvylqmaslkpedtavyycaaTTPVTSYLPRSERQYEHwgqgtqvtvss >RANKLPMP3H10,SEQ ID NO: 595; PRT; ->evqlvesggglvqsggslrlscaaagytfrARAYVMGwfrqapgkerefvaAISTGGTWTGYVDSVKDrftisrdntkntmylqmaslkpedtavyycaaTTPSTSYLPRSERQYEYwgqgtqvtvss >RANKLPMP7F9,SEQ ID NO: 596; PRT; ->evqlvesggglvqaggslrlscvasrrtfsSYAMGwfrqvpgkerdfvaAISTGSITIYGDSVKGrftisrdnakntvylqmnslkpedtavyycaaGKREPYLRQYTASNPYDYwgqgtqvtvss >RANKLPMP7E6, SEQID NO: 597; PRT; ->evqlvesggglvqaggslrlscvaskrtfaSYAMGwfrqvpgkerdfvaAITTGSITIYADSVKGrfaisrdnakntvylqmnslkpedtavyycaaGNREPYLRQYTASNPYDYwgqgtqvtvss >RANKLPMP4F4, SEQID NO: 598; PRT; ->evqlvesggglvqvgdslrlsceasgrsrfSTYVMGwfrqapgkerefvaAVSWSSGNAYYIDSAKGrfatsrdtaknimylqmnslkpedtavytcaaGRGYGLLSEYTQAPRYDYwgqgtqvtvss >RANKLPMP7B11,SEQ ID NO: 599; PRT; ->evqlvesggglvqvgdslrlsceasgrsrfSTYVMGwfrqapgkerefvaAISWSSGNAYYIDSAKGrfatsrdtaknimylqmnslkpedtavyscaaGRGYGLLSEYTQAARYDYwgqgtqvtvss >RANKLPMP9H9,SEQ ID NO: 600; PRT; ->evqlvesggglvqaggslrlscaasgrtfsRSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISSTYSESSEYDYwgqgtqvtvss >RANKLPMP9G3,SEQ ID NO: 601; PRT; ->evqlvesggglvqaggslrlscaasgrtfsRSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycavYRRTYISSTYNESSEYDYwgqgtqvtvss >RANKLPMP9E3,SEQ ID NO: 602; PRT; ->evqlvesggglvqaggslrlscaasgrtfsRSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISSTYSESSEYDYwgqgtqvtvss >RANKLPMP7H9,SEQ ID NO: 603; PRT; ->evqlvesggglvqaggslrlscaasgrtfsRSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISITYSESSDYDYwgqgtqvtvss >RANKLPMP4C3,SEQ ID NO: 604; PRT; ->evqlvesggglvqaggslrlscaasgrtfsISAMGwfrqapgkerefvcFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISSTYSESSEYDYwgqgtqvtvss >RANKLPMP9G6,SEQ ID NO: 605; PRT; ->evqlvesggglvqaggslrlscaasgrtfsRSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISSTYSESSEYDYwgqgtqvtvss >RANKLPMP7B12,SEQ ID NO: 606; PRT; ->evqlvesggglvqaggslrlscaasgrtfsRSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISSTYSESSEYDYwgqgtqvtvss >RANKLPMP7G3,SEQ ID NO: 607; PRT; ->evqlvesggglvqaggslrlscaasgrtfsRSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycavYRRTYISSTYNESSEYDYwgqgtqvtvss >RANKLPMP9C12,SEQ ID NO: 608; PRT; ->evqlvesggglvqaggslrlscaasgrtfsRSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISSTYSESSEYDYwgqgtqvtvss >RANKLPMP1D8,SEQ ID NO: 609; PRT; ->evqlvesggglvqagdslrlscaasgriftMGwfrqapgkerefvaAISGSGSITNYADSVKGrftisrdyakttvflqmnslkpedtavyycaaYVRTPYYSSYYDSTKYEYwgqgtqvtvss >RANKLPMP1A2, SEQID NO: 610; PRT; ->evqlvesggglvqagdslrlscaasgrtftMGwfrqapgkerefvaFISGSGSVTNYTDSVKGrftisrdhakntvflqmnslkpedtavyycaaYLRGPYYSSFYDSTKYEYwgqgtqvtvss >RANKLPMP1E5, SEQID NO: 611; PRT; ->evqlvesggglvqagdslrlscaasgrtftMGwfrrapgterefvaSISGSGKITNYADSVKGrftisrdhaknavflqmdglkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvss >RANKLPMP2B8, SEQID NO: 612; PRT; ->evqlvesgggsvqagdslrlscaasgrtftMGwfrqapgterefvaAISGSGKITNYADSVKGrftisrdhamntvflqmdslkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvss >RANKLPMP2C5, SEQID NO: 613; PRT; ->evqlvesggglvqagdslrlscaasgrtftMGwfrqapgterefvaAISGSGKITNYADSVKGrftisrdhakntvflqmdslkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvss >RANKLPMP2B4, SEQID NO: 614; PRT; ->evqlvesggglvqagdslrlscaasgrtftMGwfrqapgterefvaAISGSGKITNYADSVKGrftisrdhakntvflqmdslkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvss >RANKLPMP2A5, SEQID NO: 615; PRT; ->evqlvesggglvqagdslrlscaasgrtftMGwfrqapgterefvaAISGSGKITNYADSVKGrftisrdhakntvflqmdslkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvss >RANKLPMP2D7, SEQID NO: 616; PRT; ->evqlvesggglvqagdslrlscaasgrtftMGwfrqapgterefvaAISGSGKITNYADSVKGrftisrdhakntvflqmdslkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvss >RANKLPMP2G4, SEQID NO: 617; PRT; ->evqlvesggglvqagdslrlscaasgrtftMGwfrqapgterefvaAISGSGKITNYADSVKGrftisrdhakntvflqmdslkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvss >RANKLPMP7A8, SEQID NO: 618; PRT; ->emqlvesggglvqaggslrlscvaskrtfaSYAMGwfrqvpgkerdfvaAISTHSITVYADSVKGrftisrdnakntvylqmntlkpedtavyycaaGNREPYLRQYTASNPYDYwgqgtqvtvss >RANKLPMP7A5, SEQID NO: 619; PRT; ->evqlvesggglvqtggslrlscvasrrtfsSYAVGwfrqvpgkerdfvaAISTGSVTIYADSVKGrftisrdntkntvylqmnslkpedtavyycaaGNREPYLRQYTASNPYDYwgqgtqvtvss >RANKLPMP7F8, SEQID NO: 620; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLHMASLKPEDTAVYYCAATTPVTTYLPRSERQYDYWGQGTQVTVSS >RANKLPMP7F6, SEQID NO: 621; PRT; ->EVQLVESGGGLVQAGDSLRLSCAAAGFTFRRYVMGWFRQAPGKEREFVAAISTGGTWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYNCAATTPTTSYLPRSERQYEYWGQGTQVTVSS

TABLE B-2 Preferred NANOBODIES against RANK-L (non-inhibitors ofRANK/RANK-L interaction) <Name, SEQ ID #; PRT (protein); ->Sequence >RANKL3D4|051_RANKL3D4c12|3D4, SEQ ID NO: 583; PRT; ->EVQLVEAGGGLVQAGDSLRLSCAASGRTIRGTMAWFRQAPGKDREFVATVTSSGSTTFYADSVKGRFTISRDNAENTVNLQMDSLKPEDTAVYYCAARIRGKVTPSNYDYAYWGQGTQVTVSS

TABLE B-3 Bivalent NANOBODIES against RANK-L <Name, SEQ ID #; PRT(protein); -> Sequence >RANKL3-9GS-RANKL3, SEQ ID NO: 622; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL3-9GS-RANKL6, SEQ ID NO: 623;PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL3-9GS-RANKL9, SEQ ID NO: 624;PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL3-9GS-RANKL13, SEQ ID NO:625; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL3-9GS-RANKL15, SEQ ID NO:626; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL3-9GS-RANKL18, SEQ ID NO:627; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSS >RANKL6-9GS-RANKL3, SEQ ID NO:628; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL6-9GS-RANKL6, SEQ ID NO: 629;PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL6-9GS-RANKL9, SEQ ID NO:630; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL6-9GS-RANKL13, SEQ IDNO: 631; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL6-9GS-RANKL15, SEQ IDNO: 632; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL6-9GS-RANKL18, SEQ IDNO: 633; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSS >RANKL9-9GS-RANKL3, SEQ IDNO: 634; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL9-9GS-RANKL6, SEQID NO: 635; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL91biv|RANKL9-9GS-RANKL9,SEQ ID NO: 636; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL9-9GS-RANKL13, SEQID NO: 637; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL9-9GS-RANKL15, SEQID NO: 638; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL9-9GS-RANKL18, SEQID NO: 639; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSS >RANKL13-9GS-RANKL3, SEQID NO: 640; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL13-9GS-RANKL6, SEQ ID NO:641; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL13-9GS-RANKL9, SEQ IDNO: 642; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL131|RANKL13-9GS-RANKL13,SEQ ID NO: 643; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL13-9GS-RANKL15, SEQID NO: 644; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL13-9GS-RANKL18, SEQID NO: 645; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSS >RANKL15-9GS-RANKL3, SEQID NO: 646; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL15-9GS-RANKL6, SEQ ID NO:647; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL15-9GS-RANKL9, SEQ IDNO: 648; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL15-9GS-RANKL13, SEQID NO: 649; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL15-9GS-RANKL15, SEQID NO: 650; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL15-9GS-RANKL18, SEQID NO: 651; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSS >RANKL18-9GS-RANKL3, SEQID NO: 652; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL18-9GS-RANKL6, SEQ ID NO:653; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL18-9GS-RANKL9, SEQ IDNO: 654; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL18-9GS-RANKL13,SEQ ID NO: 655; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL18-9GS-RANKL15, SEQID NO: 656; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL181|RANKL18-9GS-RANKL18,SEQ ID NO: 657; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSS >RANKL3-20GS-RANKL3, SEQID NO: 658; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL3-20GS-RANKL6, SEQ IDNO: 659; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL3-20GS-RANKL9, SEQID NO: 660; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL3-20GS-RANKL13,SEQ ID NO: 661; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL3-20GS-RANKL15,SEQ ID NO: 662; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL3-20GS-RANKL18,SEQ ID NO: 663; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSS >RANKL6-20GS-RANKL3,SEQ ID NO: 664; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL6-20GS-RANKL6, SEQID NO: 665; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL6-20GS-RANKL9,SEQ ID NO: 666; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL6-20GS-RANKL13,SEQ ID NO: 667; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL6-20GS-RANKL15,SEQ ID NO: 668; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL6-20GS-RANKL18,SEQ ID NO: 669; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSS >RANKL9-20GS-RANKL3,SEQ ID NO: 670; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL9-20GS-RANKL6,SEQ ID NO: 671; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL92biv|RANKL9-20GS-RANKL9,SEQ ID NO: 672; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL9-20GS-RANKL13,SEQ ID NO: 673; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL9-20GS-RANKL15,SEQ ID NO: 674; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL9-20GS-RANKL18,SEQ ID NO: 675; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSS >RANKL13-20GS-RANKL3,SEQ ID NO: 676; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL13-20GS-RANKL6,SEQ ID NO: 677; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL13-20GS-RANKL9,SEQ ID NO: 678; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL13-20GS-RANKL13,SEQ ID NO: 679; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL13-20GS-RANKL15,SEQ ID NO: 680; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL13-20GS-RANKL18,SEQ ID NO: 681; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSS >RANKL15-20GS-RANKL3,SEQ ID NO: 682; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL15-20GS-RANKL6,SEQ ID NO: 683; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL15-20GS-RANKL9,SEQ ID NO: 684; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL15-20GS-RANKL13,SEQ ID NO: 685; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL15-20GS-RANKL15,SEQ ID NO: 686; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL15-20GS-RANKL18,SEQ ID NO: 687; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSS >RANKL18-20GS-RANKL3,SEQ ID NO: 688; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL18-20GS-RANKL6,SEQ ID NO: 689; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL18-20GS-RANKL9,SEQ ID NO: 690; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL18-20GS-RANKL13,SEQ ID NO: 691; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL18-20GS-RANKL15,SEQ ID NO: 692; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL182biv|RANKL18-20GS-RANKL18,SEQ ID NO: 693; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSRANKL133|RANKL13-30GS-RANKL13, SEQ ID NO: 766; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSRANKL183biv|RANKL18-30GS-RANKL18, SEQ ID NO: 767; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSSRANKL93biv|RANKL9-30GS-RANKL9, SEQ ID NO: 770; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSRANKL94biv|RANKL9-15GS-RANKL9, SEQ ID NO: 771; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS

TABLE B-4 Trivalent bispecific NANOBODIES against RANK-L <Name, SEQ ID#; PRT (protein); -> Sequence >RANKL30|RANKL3-ALB1-RANKL3, SEQ ID NO:694; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL3-ALB1-RANKL6, SEQ ID NO: 695; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL3-ALB1-RANKL9, SEQ ID NO: 696; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL3-ALB1-RANKL13, SEQ ID NO: 697; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL3-ALB1-RANKL15, SEQ ID NO: 698; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL3-ALB1-RANKL18, SEQ ID NO: 699; PRT; ->EVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTHISSTYSESSEYDYWGQGTQVTVSS >RANKL6-ALB1-RANKL3, SEQ ID NO: 700; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL60|RANKL6-ALB1-RANKL6, SEQ ID NO: 701; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL6-ALB1-RANKL9, SEQ ID NO: 702; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL6-ALB1-RANKL13, SEQ ID NO: 703; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL6-ALB1-RANKL15, SEQ ID NO: 704; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL6-ALB1-RANKL18, SEQ ID NO: 705; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTHISSTYSESSEYDYWGQGTQVTVSS >RANKL9-ALB1-RANKL3, SEQ ID NO: 706; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL9-ALB1-RANKL6, SEQ ID NO: 707; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL90|RANKL9-ALB1-RANKL9, SEQ ID NO: 708; PRT;->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL9-ALB1-RANKL13, SEQ ID NO: 709; PRT;->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL9-ALB1-RANKL15, SEQ ID NO: 710; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL9-ALB1-RANKL18, SEQ ID NO: 711; PRT; ->EVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTHISSTYSESSEYDYWGQGTQVTVSS >RANKL13-ALB1-RANKL3, SEQ ID NO: 712; PRT;->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL13-ALB1-RANKL6, SEQ ID NO: 713; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL13-ALB1-RANKL9, SEQ ID NO: 714; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL130|RANKL13-ALB1-RANKL13, SEQ ID NO:715; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL13-ALB1-RANKL15, SEQ ID NO: 716; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL13-ALB1-RANKL18, SEQ ID NO: 717; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTHISSTYSESSEYDYWGQGTQVTVSS >RANKL15-ALB1-RANKL3, SEQ ID NO: 718; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL15-ALB1-RANKL6, SEQ ID NO: 719; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL15-ALB1-RANKL9, SEQ ID NO: 720; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL15-ALB1-RANKL13, SEQ ID NO: 721; PRT;->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL150|RANKL15-ALB1-RANKL15, SEQ ID NO:722; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL15-ALB1-RANKL18, SEQ ID NO: 723; PRT; ->EVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTHISSTYSESSEYDYWGQGTQVTVSS >RANKL18-ALB1-RANKL3, SEQ ID NO: 724; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTHISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGKLVQAGGSLRLSCAVSGRTSSIYNMAWFRQGPGKGRESVGRIYWSDDNTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAGKTTKWSLEYDYWGQGTQVTVSS >RANKL18-ALB1-RANKL6, SEQ ID NO: 725; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTHISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKEREFVAAISPSGNTYYADSVKGRFTISRDNGKHTMYLQMNSLNPEDTAVYFCAIRATDSIYYASSYRHWGQGTQVTVSS >RANKL18-ALB1-RANKL9, SEQ ID NO: 726; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTHISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLTCAASGRTFRSYAMGWFRQAPGKEREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTQVTVSS >RANKL18-ALB1-RANKL13, SEQ ID NO: 727; PRT;->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTHISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSS >RANKL18-ALB1-RANKL15, SEQ ID NO: 728; PRT;->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTHISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAAGGTFRNYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTQVTVSS >RANKL180|RANKL18-ALB1-RANKL18, SEQ ID NO:729; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTHISSTYSESSEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSRSAMGWFRQAPGKEREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLKPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTQVTVSS

TABLE B-5 Humanized NANOBODIES against RANK-L <Name, SEQ ID #; PRT(protein); -> Sequence >RANKL6humbasic, SEQ ID NO: 730; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKGREFVAAISPSGNTYYADSVKGRFTISRDNGKHTLYLQMNSLRPEDTAVYFCAIRATDSIYYASSYRHWGQGTLVTVSS >RANKL6hum1, SEQ IDNO: 731; PRT; ->EVQLVESGGGLVQTGGSLRLSCAASGVTYSYYTASWFRQAPGKGREFVAAISPSGNTYYADSVKGRFTISRDNGKHTLYLQMNSLRPEDTAVYFCAIRATDSIYYASSYRHWGQGTLVTVSS >RANKL6hum2, SEQ IDNO: 732; PRT; ->EVQLVESGGGLMQPGGSLRLSCAASGVTYSYYTASWFRQAPGKGREFVAAISPSGNTYYADSVKGRFTISRDNGKHTLYLQMNSLRPEDTAVYFCAIRATDSIYYASSYRHWGQGTLVTVSS >RANKL6hum3, SEQ IDNO: 733; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKGREFVAAISPSGNTYYADSVKGRFTISRDNSKHTLYLQMNSLRPEDTAVYFCAIRATDSIYYASSYRHWGQGTLVTVSS >RANKL6hum4, SEQ IDNO: 734; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKGREFVAAISPSGNTYYADSVKGRFTISRDNGKNTLYLQMNSLRPEDTAVYFCAIRATDSIYYASSYRHWGQGTLVTVSS >RANKL6hum5, SEQ IDNO: 735; PRT; ->EVQLVESGGGLMQTGGSLRLSCAASGVTYSYYTASWFRQAPGKGREFVAAISPS-GNTYYADSVKGRFTISRDNGKHTLYLQMNSLRPEDTAVYFCAIRATDSIYYASSYRHWGQGTLVTVSS >RANKL9humbasic, SEQID NO: 736; PRT; ->EVQLVESGGGLVQPGGSLRLTCAASGRTFRSYAMGWFRQAPGKGREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTLVTVSS >RANKL9hum1, SEQID NO: 737; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFRSYAMGWFRQAPGKGREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTLVTVSS >RANKL9hum2, SEQID NO: 738; PRT; ->EVQLVESGGGLVQPGGSLRLTCAASGRTFRSYAMGWFRQAPGKGREFVSAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLEPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTLVTVSS >RANKL9hum3, SEQID NO: 739; PRT; ->EVQLVESGGGLVQPGGSLRLTCAASGRTFRSYAMGWFRQAPGKGREFVAAINYSGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAGSGYASLSYYSTERAYTYWGQGTLVTVSS >RANKL18humbasic,SEQ ID NO: 740; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTLVTVSS >RANKL18hum1,SEQ ID NO: 741; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYGESVKGRFTISRDNAQNTLYLQMNSLRPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTLVTVSS >RANKL18hum2,SEQ ID NO: 742; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTLVTVSS >RANKL18hum3,SEQ ID NO: 743; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTLVTVSS >RANKL18hum4,SEQ ID NO: 744; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVSFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCGVYRRTYISSTYSESSEYDYWGQGTLVTVSS >RANKL18hum5,SEQ ID NO: 745; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYGESVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSS >RANKL15humbasic,SEQ ID NO: 746; PRT; ->EVQLVESGGGLVQPGGSLRLSCAAAGGTFRNYVMGWFRQAPGKGREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTLYLQMASLRPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTLVTVSS >RANKL15hum1, SEQID NO: 747; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGGTFRNYVMGWFRQAPGKGREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTLYLQMASLRPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTLVTVSS >RANKL15hum2, SEQID NO: 748; PRT; ->EVQLVESGGGLVQPGGSLRLSCAAAGGTFRNYVMGWFRQAPGKGREFVTAISTGGSWTGYVDSVKDRFTISRDNSKNTLYLQMASLRPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTLVTVSS >RANKL15hum3, SEQID NO: 749; PRT; ->EVQLVESGGGLVQPGGSLRLSCAAAGGTFRNYVMGWFRQAPGKGREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTLYLQMNSLRPEDTAVYYCAATTPATTYLPRSERQYDYWGQGTLVTVSS >RANKL13humbasic,SEQ ID NO: 750; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFRSYPMGWFRQAPGKGREFVASITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS >RANKL13hum1, SEQID NO: 751; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFRSYPMGWFRQAPGKGREFVASITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS >RANKL13hum1, SEQID NO: 752; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYPMGWFRQAPGKGREFVASITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS >RANKL13hum2, SEQID NO: 753; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFRSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS >RANKL13hum3, SEQID NO: 754; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFRSYPMGWFRQAPGKGREFVASITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS >RANKL13hum5, SEQID NO: 755; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS >RANKL13hum5_D62E,SEQ ID NO: 756; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYAESVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS >RANKL18hum6, SEQID NO: 757; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSS >RANKL18hum7,SEQ ID NO: 765; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPLYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSS

TABLE B-6 Constructs of the humanized NANOBODIES against RANK-L <Name,SEQ ID #; PRT (protein); -> Sequence RANKL001p, SEQ ID NO: 761; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGC RANKL003p, SEQ ID NO:762; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSGGGCRANKL18hum6bi_25|RANKL18hum6-25GS-RANKL18hum6, SEQ ID NO: 768; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSRANKL18hum6bi_30|RANKL18hum6-30GS-RANKL18hum6, SEQ ID NO: 769; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSRANKL004h|RANKL13hum5-9GS-RANKL13hum5-HSA, SEQ ID NO: 772; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLRANKL006h|RANKL18hum6-30GS-RANKL18hum6-HSA, SEQ ID NO: 773; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL RANKL008a, SEQ ID NO: 759; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS RANKL010a, SEQ ID NO: 760; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSS RANKL13hum5-IgG1_1, SEQ ID NO: 774; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSGGGGSGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK RANKL13hum5-IgG1_2, SEQ ID NO: 775; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSGGGGSGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK RANKL13hum5-IgG1_3, SEQ ID NO: 776; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSGGGGSGGGSGGGGSGGGGSCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK RANKL13hum5-IgG1_4, SEQ ID NO: 777; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKRANKL13hum5-IgG2_1, SEQ ID NO: 778; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSGGGGSGGGSGGGSERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK RANKL13hum5-IgG2_2, SEQ ID NO: 779; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSGGGGSGGGSGGGGSGGGSGGSCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK RANKL13hum5-IgG2_3, SEQ ID NO: 780; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGSGGSCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK RANKL13hum5-IgG2_4, SEQ ID NO: 781; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKRANKL18hum6-IgG1_1, SEQ ID NO: 782; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSGGGGSGGGSGGGGSGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK RANKL18hum6-IgG1_2, SEQ ID NO: 783; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSGGGGSGGGSGGGGSGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK RANKL18hum6-IgG1_3, SEQ ID NO: 784; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSGGGGSGGGSGGGGSGGGSGGGGSGGGGSCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK RANKL18hum6-IgG1_4, SEQ ID NO: 785; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKRANKL18hum6-IgG2_1, SEQ ID NO: 786; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSGGGGSGGGSGGGGSGGGSGGGSERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK RANKL18hum6-IgG2_2, SEQ ID NO: 787; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSGGGGSGGGSGGGGSGGGSGGGGSGGGSGGSCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK RANKL18hum6-IgG2_3, SEQ ID NO: 788; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGSGGSCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK RANKL18hum6-IgG2_4, SEQ ID NO: 789; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSRSAMGWFRQAPGKGREFVGFITGSGGTTYYADSVKGRFTISRDNAQNPVYLQMNSLRPEDTAVYYCAVYRRTYISSTYSESSEYDYWGQGTLVTVSSCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKCabLys3-A1b11-13H5, SEQ ID NO: 815; PRT; ->DVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS 13H5-CabLys3-13H5, SEQ ID NO: 816; PRT;->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLV

TABLE B-7 NANOBODIES against human serum albumin SEQ ID Name Sequence NOALB-1 EVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQA 790PGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS ALB-12EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQA 791PGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS

TABLE B-8 Linkers used in the NANOBODY constructs SEQ ID Linker SequenceNO 9GS linker GGGGSGGGS 792 15GS linker ggggsggggsggggs 793 20GS linkerGGGGSGGGGSGGGGSGGGGS 794 25GS linker ggggsggggsggggsggggsggggs 795 30GSlinker GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 796

TABLE C-1 Anti-RANK-L NANOBODY sequences obtained as described inExample 1. The NANOBODY sequences are grouped by family. Uniquesequences are indicated UNIQUE SEQUENCES RANKLPMP2G8 (RANKL3)(SEQ ID NO:562)        evqlvesggklvqaggslrlscavsgrtss--IYNMAwfrqgpgkgresvgRIYWSDDNTYYADSVKGrftisrdnatntvylqmnslkpedtavyycagKTTKWSLEYDY-------wgqgtqvtvssRANKLPMP2B9 (RANKL2)(SEQ ID NO:561)        evqlvesggglvqpggslrlscaasgftfs--SYYMSwvrqapgkglewvsSIYSDGSTTDYADSVKGrftisrdnakntlnlqmnslksedtavyycakDANSGGLEYDY-------wgqgtqvtvssRANKLPMP4H9 (RANKL6)(SEQ ID NO:565)        evqlvesggglmqtggslrlscaasgvtys--YYTASwfrqapgkerefvaAISPSG-NTYYADSVKGrftisrdngkhtmylqmnslnpedtavyfcaiRATDSIYYASSYRH----wgqgtqvtvssRANKLPMP4G8 (RANKL5)(SEQ ID NO:564)        evqlvesggglvqaggslrlscaasgrtig--GHTMAwfrqapgkerdfvaTITSSGSTIFYADSVKGrftisrdngkktmtlemdslkpedtavyycaaRIRGKVTVDYFDYAY---wgqgtqvtvssRANKLPMP7B4 (RANKL7)(SEQ ID NO:566)        evqlveseggpvqsggslrlscaasgrtfs--vstiawfrqapgegrefvaaiypsgrnayvadsvkgrftisrdnakktvylqmnslkpedtaayycaahqpsgsyysaeayay---wgqgtqvtvssRANKLPMP9F10 (RANKL11)(SEQ ID NO:570)      evqlvesggglvqaggslrlscaasgrtfs--SKTMGwfrqppgnerefvaAITPTSRTTYYADSVKGrftisrdnakntvslqmnslkfedtaayycvaVRRYGSPPHDGSSYEY--wgqgtqvtvssRANKLPMP9B3 (RANKL10)(SEQ ID NO:569)        evqlvesggglvqaggslrlscaasgitfs--SRTMGwfrqapgkerefvaAITPSSRTTYYADSVKGrftisrdnakntvllqmnslkpedtavyycaaERTYGSNYTRPTAWNY--wgqgtqvtvssRANKLPMP4F12 (RANKL4)(SEQ ID NO:563)        kvqlvesggglvqtgdslrlscaasgraig--SYAMGwfrqapgkerefvaVINYRGSSLKYADRVKGrftisrdnaknmvylqmnslkpddtavyycaaQTSGADFGTTPQRYTY--wgqgtqvtvssRANKLPMP9B1 (RANKL1)(SEQ ID NO:560)        evqlvesggglvqaggslrlscavsgrtfs--SSTMAwfrqppggerdfvaSISTSGTRTLYADSVKGrftisrdnakstgylqmnslkpedtavyfcaaVNRRGWEFWRLASGYDY-wglgaqvtvssRANKLPMP7E3 (RANKL8)(SEQ ID NO:567)        evqlvesgggsvqpggslrlscaasggtfs--RYAMGwfrqapgkerefvsAISVGGTYQYYVDSVKGrftisrdnaestvylqmnslkpedtavyycagDASPYGYLREYTATRFDYwgqgtqvtvssRANKLPMP8A11 (RANKL9)(SEQ ID NO:568)        evqlvesggglvqaggslrltcaasgrtfr--SYAMGwfrqapgkerefvaAINYSGGSTNYADSVKGrftisrdnakntlylqmnslepedtavyycaaGSGYASLSYYSTERAYTYwgqgtqvtvssFAMILIES FAMILY 1 RANKLPMP9B6 (RANKL12)(SEQ ID NO:571)       evqlvesgggwmqaggslrlscaasgrtft-----MAwfrqapgkerefvaAITGSGRSTYYTDSVKGrftisrdnakntaylqmkslkpedtavyycagLRGLG-LEYDSAKS-YSYwgqgtqvtvssRANKLPMP4B3 (SEQ ID NO: 584)     evqlvesgggwmqaggslrlscaasgrtft-----MAwfrqasgkerefvaAITGSGRSTYYTDSVKGrftisrdnakntaylqmkslkpedtavyycagLRGLG-LEYDSAKS-YSYwgqgtqvtvssFAMILY 2 RANKLPMP1C7 (RANKL13)(SEQ ID NO:572)       evqlvesggglvqaggslrlscaasgrtfr--SYPMGwfrqapgkerefvaSITGSGGSTYYADSVKGrftisrdnakntvylqmnslrpedtavyscaaYIRPDTYLSRDYRKYDY-wgqgtqvtvssRANKLPMP2E11 (SEQ ID NO: 585)    EV*LVESGGGLVQAGGSLRLSCAASGRTFR--SYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDY-WGQGTQVTVSSFAMILY 3 RANKLPMP2A6 (SEQ ID NO:586)     evqlvesggglvqaggslrlscaasgltss--RYTMSwfrqdpgkerefvaAVPLSG-NTYYADPVRGrftisrdnakntvdlqmnslkpedtavyycaaRASGSIFNRGS----YAYwgqgtqvtvssRANKLPMP6B8 (RANKL14)(SEQ ID NO:573)       evqlvesggglvqaggslrlscaasgrtss--YYTMSwfrqdpgkerefvaAVPLSG-NTYYADPVRGrftisrdnakntadlqmnslkpedtavyycaaRASGSIYNRGS----YAYwgqgtqvtvssRANKLPMP1F2 (SEQ ID NO:587)     evqlvesggglvpaggslrlscaasgltdr--RYTMSwfrqdpgkerefvaAVPLSG-NTYYADPVRGrftisrdnakntvdlqmnslkpedtavyycaaRASGSIFNRGS----YAYwgqgtqvtvssRANKLPMP2D4 (SEQ ID NO:588)     evqlvesggglvpaggslrlscaasgltdr--RYTMSwfrqdpgkerefvaAVPLSG-NTYYADPVRGrftisrdnakntvdlqmnslkpedtavyycaaRASGSIFNRGS----YAYwgqgtqvtvssFAMILY 4 RANKLPMP7B2 (SEQ ID NO:589)     evqlvesggglvqaggslrlscaaaggtfr--NYVMGwfrqapgkerefvtAISTGGSWTGYVDSVKDrftisrdntkntvylqmaslkpedtavyycaaTMPATTYLPR-SERQYDYwgqgtqvtvssRANKLPMP7C5 (RANKL15)(SEQ ID NO:574)       evqlvesggglvqaggslrlscaaaggtfr--NYVMGwfrqapgkerefvtAISTGGSWTGYVDSVKDrftisrdntkntvylqmaslkpedtavyycaaTTPATTYLPR-SERQYDYwgqgtqvtvssRANKLPMP7A11 (SEQ ID NO: 590)    evqlvesggglvqaggsltlscaaagftfr--RYVMGwfrqapgkerefvaAISTGGTWTGYVDSVKDrftisrdntkntvylqmaslkpedtavyncaaTTPTTSYLPR-SERQYEYwgqgtqvtvssRANKLPMP7F1 (SEQ ID NO: 591)     evqlvesggglvqaggslrlscaaagctfr--NYVMGwfrqapgkerefvtAISTGGTWTGYVDSVKDrftisrdntkntvnlqmaslkpedtavyycaaTTPTTSYLPR-SERQYEYwgqgtqvtvssRANKLPMP7H5 (SEQ ID NO: 592)     evqlvesggglvqaggslrlscaaaggtfr--NYVMGwfrqapgkerefvaAISTGGSWTGYVDSVKDrftisrdntkntvylqmvslkpedtavyycaaTTPATTYLPR-SERQYDYwgqgtqvtvssRANKLPMP7E7 (SEQ ID NO: 593)     evqlvesggglvqaggslrlscaaaggtfr--NYVMGwfrqapgkerefvtAISAGGSWTGYVDSVKDrftisrdntkntvylqmaslkpedtavyycaaTTPATTYLPR-SERQYDYwgqgtqvtvssRANKLPMP7E2 (SEQ ID NO: 594)     evqlvesggglvqaggslrlscaaagytfr--AYVMGwfrqapgkerefvaGISTGGTWTGYVDSVKDrftisrdntkntvylqmaslkpedtavyycaaTTPVTSYLPR-SERQYEHwgqgtqvtvssRANKLPMP3H10 (SEQ ID NO:595)    evqlvesggglvqsggslrlscaaagytfrARAYVMGwfrqapgkerefvaAISTGGTWTGYVDSVKDrftisrdntkntmylqmaslkpedtavyycaaTTPSTSYLPR-SERQYEYwgqgtqvtvssRANKLPMP7F8 (SEQ ID NO: 620)     EVQLVESGGGLVQAGGSLRLSCAAAGGTFR--NYVMGWFRQAPGKEREFVTAISTGGSWTGYVDSVKDRFTISRDNTKNTVYLHMASLKPEDTAVYYCAATTPVTTYLPR-SERQYDYWGQGTQVTVSSRANKLPMP7F6 (SEQ ID NO: 621)     EVQLVESGGGLVQAGDSLRLSCAAAGFTFR--RYVMGWFRQAPGKEREFVAAISTGGTWTGYVDSVKDRFTISRDNTKNTVYLQMASLKPEDTAVYNCAATTPTTSYLPR-SERQYEYWGQGTQVTVSSFAMILY 5 RANKLPMP7G8 (RANKL16)(SEQ ID NO:575)       evqlvesggglvqaggslrlscvasrrtfs--SYAMGwfrqvpgkerdfvaAISTG-SITIYGDSVKGrftisrdnakntvylqmnslkpedtavyycaaGKREPYLRQYTASNPYDYwgqgtqvtvssRANKLPMP7F9 (SEQ ID NO:596)     evqlvesggglvqaggslrlscvasrrtfs--SYAMGwfrqvpgkerdfvaAISTG-SITIYGDSVKGrftisrdnakntvylqmnslkpedtavyycaaGKREPYLRQYTASNPYDYwgqgtqvtvssRANKLPMP7E6 (SEQ ID NO:597)     evqlvesggglvqaggslrlscvaskrtfa--SYAMGwfrqvpgkerdfvaAITTG-SITIYADSVKGrfaisrdnakntvylqmnslkpedtavyycaaGNREPYLRQYTASNPYDYwgqgtqvtvssFAMILY 6 RANKLPMP4F4 (SEQ ID NO:598)     evqlvesggglvqvgdslrlsceasgrsrf-STYVMGwfrqapgkerefvaAVSWSSGNAYYIDSAKGrfatsrdtaknimylqmnslkpedtavytcaaGRGYGLLSEYTQAPRYDYwgqgtqvtvssRANKLPMP9C2 (RANKL17)(SEQ ID NO:576)       evqlvesggglvqvgdslrlsceasgrsrf-STYVMGwfrqapgkerefvaAVSWSSGNAYYIDSAKGrfatsrdtaknimylqmnslkpedtavytcaaGRGYGLLSEYTQAPRYDYwgqgtqvtvssRANKLPMP7B11 (SEQ ID NO: 599)    evqlvesggglvqvgdslrlsceasgrsrf-STYVMGwfrqapgkerefvaAISWSSGNAYYIDSAKGrfatsrdtaknimylqmnslkpedtavyscaaGRGYGLLSEYTQAARYDYwgqgtqvtvssFAMILY 7 RANKLPMP9H9 (SEQ ID NO:600)     evqlvesggglvqaggslrlscaasgrtfs--RSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISSTYSESSEYDYwgqgtqvtvssRANKLPMP9G3 (SEQ ID NO: 601)     evqlvesggglvqaggslrlscaasgrtfs--RSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycavYRRTYISSTYNESSEYDYwgqgtqvtvssRANKLPMP9E3 (SEQ ID NO: 602)     evqlvesggglvqaggslrlscaasgrtfs--RSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISSTYSESSEYDYwgqgtqvtvssRANKLPMP7H9 (SEQ ID NO: 603)     evqlvesggglvqaggslrlscaasgrtfs--RSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISITYSESSDYDYwgqgtqvtvssRANKLPMP4C3 (SEQ ID NO: 604)     evqlvesggglvqaggslrlscaasgrtfs--ISAMGwfrqapgkerefvcFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISSTYSESSEYDYwgqgtqvtvssRANKLPMP9G6 (SEQ ID NO: 605)     evqlvesggglvqaggslrlscaasgrtfs--RSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISSTYSESSEYDYwgqgtqvtvssRANKLPMP7F11 (RANKL18)(SEQ ID NO:577)      evqlvesggglvqaggslrlscaasgrtfs--RSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISSTYSESSEYDYwgqgtqvtvssRANKLPMP7B12 (SEQ ID NO: 606)    evqlvesggglvqaggslrlscaasgrtfs--RSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISSTYSESSEYDYwgqgtqvtvssRANKLPMP7G3 (SEQ ID NO: 607)     evqlvesggglvqaggslrlscaasgrtfs--RSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycavYRRTYISSTYNESSEYDYwgqgtqvtvssRANKLPMP9C12 (SEQ ID NO: 608)    evqlvesggglvqaggslrlscaasgrtfs--RSAMGwfrqapgkerefvgFITGSGGTTYYGESVKGrftisrdnaqnpvylqmnslkpedtavyycgvYRRTYISSTYSESSEYDYwgqgtqvtvssFAMILY 8 RANKLPMP1D8 (SEQ ID NO:609)     evqlvesggglvqagdslrlscaasgrift-----MGwfrqapgkerefvaAISGSGSITNYADSVKGrftisrdyakttvflqmnslkpedtavyycaaYVRTPYYSSYYDSTKYEYwgqgtqvtvssRANKLPMP1A2 (SEQ ID NO: 610)     evqlvesggglvqagdslrlscaasgrtft-----MGwfrqapgkerefvaFISGSGSVTNYTDSVKGrftisrdhakntvflqmnslkpedtavyycaaYLRGPYYSSFYDSTKYEYwgqgtqvtvssRANKLPMP6C8 (RANKL19)(SEQ ID NO:578)       evqlvesggglvqagdslrlscaasgrtvt-----MGwfrqapgkerefvaSITGSGSVTNYADSVKGrftisrdnakntvflqmnslkpedtavyycaaYLPSPYYSSYYDSTKYEYwgqgtqvtvssRANKLPMP1E5 (SEQ ID NO: 611)     evqlvesggglvqagdslrlscaasgrtft-----MGwfrrapgterefvaSISGSGKITNYADSVKGrftisrdhaknavflqmdglkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvssRANKLPMP2F4 (RANKL20)(SEQ ID NO:579)       evqlvesggglvqagdslrlscaasgrtft-----MGwfrqapgterefvaAISGSGKITNYADSVKGrftisrdhakntvflqmdslkpedtavyycagYLRSPYYSSFYDSAKYEYwgqgtqvtvssRANKLPMP2B8 (SEQ ID NO: 612)     evqlvesgggsvqagdslrlscaasgrtft-----MGwfrqapgterefvaAISGSGKITNYADSVKGrftisrdhamntvflqmdslkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvssRANKLPMP2C5 (SEQ ID NO: 613)     evqlvesggglvqagdslrlscaasgrtft-----MGwfrqapgterefvaAISGSGKITNYADSVKGrftisrdhakntvflqmdslkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvssRANKLPMP2B4 (SEQ ID NO: 614)     evqlvesggglvqagdslrlscaasgrtft-----MGwfrqapgterefvaAISGSGKITNYADSVKGrftisrdhakntvflqmdslkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvssRANKLPMP2A5 (SEQ ID NO: 615)     evqlvesggglvqagdslrlscaasgrtft-----MGwfrqapgterefvaAISGSGKITNYADSVKGrftisrdhakntvflqmdslkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvssRANKLPMP2D7 (SEQ ID NO: 616)     evqlvesggglvqagdslrlscaasgrtft-----MGwfrqapgterefvaAISGSGKITNYADSVKGrftisrdhakntvflqmdslkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvssRANKLPMP2G4 (SEQ ID NO: 617)     evqlvesggglvqagdslrlscaasgrtft-----MGwfrqapgterefvaAISGSGKITNYADSVKGrftisrdhakntvflqmdslkpedtavyycaaYLRSPYYSSYYDSAKYEYwgqgtqvtvssFAMILY 9 RANKLPMP7A8 (SEQ ID NO:618)     emqlvesggglvqaggslrlscvaskrtfa--SYAMGwfrqvpgkerdfvaAISTH-SITVYADSVKGrftisrdnakntvylqmntlkpedtavyycaaGNREPYLRQYTASNPYDYwgqgtqvtvssRANKLPMP7C6 (RANKL21)(SEQ ID NO:580)       evqlvesggglvqaggslrlscvasrrtfn--SYAVGwfrqvpgeerdfvaAISTG-SVTIYADSVKGrftisrdnakntvylqmnslkpedtavyycaaGNREPYLRQYTASNPYDYwgqgtqvtvssRANKLPMP7A5 (SEQ ID NO:619)     evqlvesggglvqtggslrlscvasrrtfs--SYAVGwfrqvpgkerdfvaAISTG-SVTIYADSVKGrftisrdntkntvylqmnslkpedtavyycaaGNREPYLRQYTASNPYDYwgqgtqvtvss

TABLE C-2 ED50 values obtained with the anti-RANK-L NANOBODIES in ELISAED50 NANOBODY (pM) RANKL1 619 RANKL3 568 RANKL4 380 RANKL6 385 RANKL7376 RANKL8 652 RANKL9 542 RANKL11 517 RANKL12 399 RANKL13 226 RANKL14574 RANKL15 266 RANKL16 953 RANKL17 453 RANKL18 242 RANKL19 506 RANKL20306 RANKL21 861 RANKL22 379 RANKL23 679

TABLE C-3 IC50 values obtained with the anti-RANK-L NANOBODIES inAlphaScreen assay IC50 NANOBODY (pM) RANKL1 739 RANKL3 497 RANKL4 582RANKL6 288 RANKL7 459 RANKL8 755 RANKL9 137 RANKL11 1170 RANKL12 627RANKL13 185 RANKL14 1750 RANKL15 283 RANKL16 2250 RANKL17 737 RANKL18146 RANKL19 2240 RANKL20 1360 RANKL21 1580 RANKL22 3020 RANKL23 3530

TABLE C-4 Dose-dependent binding of NANOBODIES to membrane-bound humanand cyno RANK-L (PE mean fluorescence) NANOBODY human RANK-L cynomolgusRANK-L (pM) RANKL6 RANKL9 RANKL13 RANKL15 RANKL18 irrelevant RANKL13RANKL15 RANKL18 irrelevant 2000 12752 4865 13918 7724 7633 87 1244210956 12032 149 400 4579 3365 5225 2641 2327 90 4107 3410 3488 140 801697 2279 1534 715 649 82 16 434 1350 499 250 218 77 3.2 347 381 220 124105 84

TABLE C-5 Comparison of monovalent and trivalent bispecific anti-RANK-LNANOBODIES in AlphaScreen IC50 NANOBODY (pM) RANKL3 474 RANKL6 777RANKL9 341 RANKL13 147 RANKL15 572 RANKL18 187 RANKL30 44 RANKL60 26RANKL90 68 RANKL130 29 RANKL150 92 RANKL180 33

TABLE C-6 Comparison of monovalent and trivalent bispecific anti-RANK-LNANOBODIES in cell based competitive binding assay NANOBODY IC50 (pM)RANKL13 429 RANKL15 534 RANKL18 552 RANKL130 55 RANKL150 210 RANKL180106

TABLE C-7 Potency of anti-RANK-L NANOBODIES in NF-kappaB reporter geneassay Compound IC50 (nM) Compound IC50 (nM) RANKL13 6.205 ± 3.019RANKL130 2.375 ± 2.379 (n = 2) (n = 2) RANKL15 10.03 ± 2.390 RANKL1506.24 ± 4.765 (n = 2) (n = 2) RANKL18 15.24 ± 1.923 RANKL180 3.95 ± 0.183(n = 2) (n = 2) RANKL3 8.32 ± 3.747 OPG 6.03 (n = 2) (n = 1)

TABLE C-8 Primers used for the construction of the human/ mouse hybridsas described in Example 6. SEQ ID Name Sequence 5′ to 3′ NO T7-EEVAAGGCTAGAGTACTTAATACGA 799 RevpCIneo CTTATCATGTCTGCTCGAAGC 800RevhRANKLM1 GGGAACCAGATGGGATGGAGGCGGCATTAATAGTGA 801 GATGAG FwhRANKLM1CTCATCTCACTATTAATGCCGCCTCCATCCCATCTG 802 GTTCCC RevhRANKLM2CCATTAGTTGAAGATACTCTGTAGCCACGCTTCCTG 803 AAGTTTCATG FwhRANKLM2CATGAAACTTCAGGAAGCGTGGCTACAGAGTATCTT 804 CAACTAATGG RevhRANKLM3CCATTAGTTGAAGATAGTCTGTAGGTAGGTCTCC 805 FwhRANKLM3GGAGACCTACCTACAGACTATCTTCAACTAATGG 806

TABLE C-9 Binding of RANKL NANOBODIES on human/mouse hybrids of RANK-Las described in Example 6. RANK-L NANOBODY hRANK-L h/m hybrid 1 h/mhybrid 2 h/m hybrid 3 RANKL3 + + + + RANKL6 + + − + RANKL9 + + + −RANKL13 + + − − RANKL15 + + − − RANKL18 + + + − MoAb¹ + + + +¹Monoclonal antibody against human RANK-L (R&D Systems, Minneapolis, MN;Cat No. 6262).

TABLE C-10 Dosing of the animals in the pharmacokinetics andpharmacodynamics study as described in Example 7. Dose and route GroupAnimal ID of administration Single/Repeated RANKL130 1m 10 mg/kg (IVbolus) Single 2f 10 mg/kg (IV bolus) Single RANKL131 3m 15 mg/kg(infusion 8 hrs) Single 4f 15 mg/kg (infusion 8 hrs) Single RANKL180 5m10 mg/kg (IV bolus) Single 6f 10 mg/kg (IV bolus) Single ALB1 7m 10mg/kg (IV bolus) Single 8f 10 mg/kg (IV bolus) Single Ibandronate 9m0.15 mg/kg (IV bolus) Repeated (monthly) 10f 0.15 mg/kg (IV bolus)Repeated (monthly)

TABLE C-11 Values obtained for the pharmacokinetic parameters in theCynomolgus Monkeys study as described in Example 7. Parameter¹ cyno 1mcyno 2f cyno 5m cyno 6f V_(ss) _(—) _(pred) (mL/kg) 67.7 71.1 67.1 55.4CL _(—) _(pred) (mL/day/kg) 7.71 7.91 5.86 5.08 MRT_(inf) _(—) _(pred)(day) 8.79 8.98 11.5 10.9 t_(1/2 λz1) (day)² 6.75 6.81 8.74 9.28 λ_(z1)_(—) _(lower) − λ_(z1) _(—) _(upper)  1-39  2-51  4-51  1-32 (day) R²t_(1/2 λz1) 0.992 0.998 0.993 0.982 t_(1/2 λz2) (day)³ 3.10 3.29 2.532.21 λ_(z2) _(—) _(lower) − λ_(z2) _(—) _(upper) 39-58 51-72 58-72 36-51(day) R² t_(1/2 λz2) 0.994 0.985 0.991 0.984 AUC_(0-39 days) 1287 — 1693— AUC_(0-51 days) — 1260 — 1909 AUC_(last) (μg · day/mL) 1297 1264 17071970 % AUC_(extrap) _(—) _(pred) 0.0117 0.0033 0.0046 0.0112 AUC_(inf)_(—) _(pred) (μg · 1297 1264 1708 1970 day/mL) AUC_(inf) _(—) _(pred)/D(kg · 0.130 0.126 0.171 0.197 day/mL) ¹All parameters were calculatedwith non-compartmental modelling. ²Half-life calculated on apparent betaphase. ³Half-life calculated on apparent gamma phase.

TABLE C-12 Values obtained for the pharmacokinetic parameters in theCynomolgus Monkeys study as described in Example 7. Parameter¹ cyno 3mcyno 4f V_(ss) _(—) _(pred) (mL/kg) 488 775 CL _(—) _(pred) (mL/day/kg)2433 3355 MRT_(inf) _(—) _(pred) (day) 0.200 0.231 t_(1/2 λz) (day)0.787 0.843 λ_(z1) _(—) _(lower) − λ_(z1) _(—) _(upper) (day) 1.0-4.01.0-4.0 R² t_(1/2 λz) 0.970 0.918 AUC_(last) (μg · day/mL) 6.14 4.45 %AUC_(extrap) _(—) _(pred) 0.380 0.493 AUC_(inf) _(—) _(pred) (μg ·day/mL) 6.16 4.47 AUC_(inf) _(—) _(pred)/D (kg · day/mL) 0.0004 0.0003¹All parameters calculated with non-compartmental modelling

TABLE C-13 Activity of the humanized RANKL NANOBODIES in AlphaScreen andBIACORE 3000. AlphaScreen BIACORE NANOBODY IC50 (pM) Kd (s−1) Ka(M−1s−1) KD (M) RANKL13 174 2.50E−04 5.00E+05 5.00E−10 RANKL13basic 1591.89E−04 8.85E+05 2.14E−10 RANKL13hum1 123 1.45E−04 1.37E+06 1.06E−10RANKL13hum2 191 2.32E−04 2.64E+05 8.80E−10 RANKL13hum3 196 1.74E−046.59E+05 2.64E−10 RANKL13hum4 223 2.04E−04 3.79E+05 5.38E−10 RANKL13hum591 2.10E−04 8.00E+05 2.60E−10 RANKL18 292 5.60E−05 3.10E+05 1.80E−10RANKL18basic 219 1.00E−04 6.12E+05 1.63E−10 RANKL18hum1 3153 2.36E−041.51E+05 1.56E−09 RANKL18hum2 3186 1.73E−04 6.34E+04 2.73E−09RANKL18hum3 274 1.27E−04 4.41E+05 2.88E−10 RANKL18hum4 ND ND ND NDRANKL18hum5 253 9.94E−05 4.29E+05 2.32E−10 RANKL18hum6 138 6.72E−055.00E+05 1.60E−10 RANKL18hum7 572 2.70E−04 1.80E−05 1.50E−09

TABLE C-14 Activity of different NANOBODY constructs in AlphaScreen andFMAT. AlphaScreen FMAT NANOBODY IC50(pM) IC50(pM) RANKL13 120 264RANKL130 31 45 RANKL131 19 23 RANKL133 26 ND RANKL18 253 253 RANKL180 3956 RANKL181biv 113 134 RANKL182biv 89 76 RANKL183biv 49 52RANKL18hum6Bi_25 100 38 RANKL18hum6Bi_30 50 38 RANKL9 238 130 RANKL90 6349 RANKL91biv 153 90 RANKL92biv 70 32 RANKL93biv 66 48 RANKL94biv 72 30

TABLE C-15 Different construct made of the humanized NANOBODIES.NANOBODY SEQ NANOBODY format ID ID NO RANKL13hum5-ALB012-RANKL13hum5RANKL008a 759 RANKL18hum6-ALB012-RANKL18hum6 RANKL010a 760RANKL13hum5-9GS-RANKL13hum5-PEG RANKL001p 761RANKL18hum6-30GS-RANKL18hum6-PEG RANKL003p 762RANKL13hum5-9GS-RANKL13hum5-HSA RANKL004h 772RANKL18hum6-30GS-RANKL18hum6-HSA RANKL006h 773

TABLE C-16 Activity of the different constructs of the humanizedNANOBODIES in AlphaScreen and FMAT. AlphaScreen FMAT NANOBODY IC50 (pM)IC50 (pM) RANKL13hum5 188 137 RANKL008a 25 36 RANKL001p 49 41 RANKL004h48 34 RANKL130 17 42 RANKL18hum6 87 380 RANKL3p 25 38 RANKL10a 28 57RANKL180 30 NA

TABLE C-17 Basic pharmacokinetic parameters of NANOBODIES after a singleintravenous administration (100 μg/animal) in the male Balb/c mouse.RANKL008a RANKL010a RANKL001p RANKL003p Parameter Mean SD CV (%) Mean SDCV (%) Mean SD CV (%) Mean SD CV (%) C(0) (g/ml) 87.2 8.2 9 80.8 25.5 32— — — 122 61 50 Vss (mL) 2.03 0.05 2 2.73 0.72 26 — — — 1.32 0.61 46 Vc(mL) 1.16 0.11 9 1.35 0.52 39 — — — 1.08 0.77 71 Vt (mL) 0.870 0.158 181.38 0.21 15 — — — 0.250 0.159 64 CL (mL/day) 1.15 0.08 7 1.36 0.40 29 —— — 0.629 0.263 42 CLd (mL/day) 2.06 0.18 9 5.12 0.95 19 — — — 1.09 0.4138 t 1/2 alpha (hr) 3.55 0.35 10 2.06 0.48 23 — — — 2.99 2.41 81 t 1/2beta (day) 1.37 0.01 1 1.50 0.09 6 — — — 1.53 0.07 4 MRT (day) 1.77 0.084 2.02 0.10 5 — — — 2.08 0.09 4 AUCinf (g″day/ml) 87.4 6.2 7 77.1 19.125 — — — 176 60 34 AUCinf/D (day/ml) 0.874 0.062 7 0.771 0.191 25 — — —1.76 0.60 34

TABLE C-18 Basic pharmacokinetic parameters of NANOBODIES after a singlesubcutaneous administration (100 μg/animal) in the male Balb/c mouse.RANKL008a RANKL010a RANKL001p RANKL003p Parameter Mean SD CV (%) Mean SDCV (%) Mean SD CV (%) Mean SD CV (%) V/F (mL) 2.59 0.34 13 2.39 0.10 44.53 4.68 103 1.01 0.29 29 CL/F (mL/hr) 1.41 0.19 13 1.12 0.01 1 1.932.03 105 0.447 0.109 24 tlag (min) — — — — 81.9 68.5 83 — — t 1/2absorption (hr) 20.4 6.8 33 13.6 0.4 3 7.8 1.5 19 12.2 2.8 23 t 1/2elimination (day) 1.27 0.04 3 1.49 0.06 4 1.65 0.04 2 1.55 0.08  5 tmax(day) 1.47 0.24 16 1.27 0.04 3 1.00 0.09 9 1.21 0.14 12 Cmax (μg/ml)17.5 1.5 9 23.1 0.7 3 27.1 18.0 66 60.6 14.3 24 AUCinf (μg · hr/ml) 72.09.4 13 89.7 0.9 1 95.8 62.8 66 233 59 25 AUCinf/D (hr/ml) 0.720 0.094 130.897 0.009 1 0.958 0.628 66 2.33 0.59 25 F (%) 82.4 102 — 110-133

TABLE C-19 Dose, route and frequency of administration used in animalmodel as described in Example 14. Dose Route/ Animal Group Test ItemLevel frequency admin. numbers 1 RANKL008a 3.0 IV/Once 1-3 2 RANKL008a0.3 IV/Once 4-6 3 RANKL008a 0.03 IV/Once 7-9 4 RANKL008a 3.0 SC/Once10-12 5 RANKL001p 3.0 IV/Once 13-15 6 RANKL001p 0.3 IV/Once 16-18 7RANKL001p 0.03 IV/Once 19-21 8 RANKL001p 3.0 SC/Once 22-24 9 RANKL003p3.0 IV/Once 25-27 10 RANKL003p 0.3 IV/Once 28-30 11 RANKL003p 0.03IV/Once 31-33 12 RANKL003p 3.0 SC/Once 34-36 13 RANKL130 10 IV/Once37-39 14 RANKL130 3.0 IV/Once 40-42 15 Ibandronate 0.15 IV/Every Month43-45 16 ALB8 3.0 IV/Once 46-48 17 PBS IV/Once 49-51

TABLE C-20 Basic pharmacokinetic parameters of RANKL008a after a singleintravenous bolus administration in the female cynomolgus monkey asdescribed in Example 14. 3 mg/kg 0.3 mg/kg 0.03 mg/kg parameter Mean SDMean SD Mean SD Vss_pred (mL/kg) 65.8 4.3 74.2 12.8 85.3 7.9 CL_pred(mL/day/kg) 8.33 1.71 14.0 0.6 50.8 11.0 MRTinf_pred (day) 8.07 1.375.29 0.68 1.72 0.31 t 1/2 λz1 (day)¹ 7.28 0.07 5.83 0.27 — — λz1_lower(day) — — — — — — λz1_upper (day) — — — — — — R2 t 1/2 λz1 — — — — — — t1/2 λz2 (day)² 1.65 1.42 1.28 0.58 1.18 0.20 λz2_lower (day) — — — — — —λz2_upper (day) — — — — — — R2 t 1/2 λz2 — — — — — — AUClast (mg ·day/ml) 370 74 19.5 3.0 0.546 0.186 % AUCextrap_pred 0.0510 0.0584 2.103.26 11.7 14.2 AUCinf_pred (mg · day/ml) 370 74 20.0 3.4 0.609 0.140AUCinf_pred/D (kg · day/ml) 0.124 0.025 0.0716 0.0034 0.0203 0.0047¹Half-life calculated on apparent beta phase ²Half-life calculated onapparent gamma phase

TABLE C-21 Basic pharmacokinetic parameters of RANKL001p after a singleintravenous bolus administration in the female cynomolgus monkey asdescribed in Example 14. 3 mg/kg 0.3 mg/kg 0.03 mg/kg parameter Mean SDMean SD Mean SD Vss_pred (mL/kg) 60.4 4.6 73.3 4.3 67.5 0.9 CL_pred(mL/day/kg) 8.69 0.82 15.0 1.5 41.3 4.5 MRTinf_pred (day) 6.95 0.13 4.900.51 1.64 0.18 t 1/2 λz1 (day)¹ 5.14 0.15 4.11 0.44 — — λz1_lower (day)— — — — — — λz1_upper (day) — — — — — — R2 t 1/2 λz1 — — — — — — t 1/2λz2 (day)² 1.28 0.63 1.95 0.39 1.11 0.09 λz2_lower (day) — — — — — —λz2_upper (day) — — — — — — R2 t 1/2 λz2 — — — — — — AUClast (mg ·day/ml) 346 31 19.7 2.0 0.670 0.095 % AUCextrap_pred 0.0191 0.0072 1.651.18 3.95 1.97 AUCinf_pred (mg · day/ml) 346 31 20.0 1.9 0.697 0.092AUCinf_pred/D (kg · day/ml) 0.115 0.010 0.0669 0.0065 0.0244 0.0026¹Half-life calculated on apparent beta phase ²Half-life calculated onapparent gamma phase

TABLE C-22 Basic pharmacokinetic parameters of RANKL003p after a singleintravenous bolus administration in the female cynomolgus monkey asdescribed in Example 14. 3 mg/kg 0.3 mg/kg 0.03 mg/kg parameter Mean SDMean SD Mean SD Vss_pred (mL/kg) 33.1 1.8 31.1 3.9 32.5 5.9 CL_pred(mL/day/kg) 4.49 0.04 5.93 0.60 14.9 2.9 MRTinf_pred (day) 7.38 0.345.25 0.50 2.21 0.37 t 1/2 λz1 (day)¹ 5.75 1.10 4.57 0.24 — — λz1_lower(day) — — — — — — λz1_upper (day) — — — — — — R2 t 1/2 λz1 — — — — — — t1/2 λz2 (day)² 1.39 0.12 2.06 0.37 1.52 0.24 λz2_lower (day) — — — — — —λz2_upper (day) — — — — — — R2 t 1/2 λz2 — — — — — — AUClast (mg ·day/ml) 647 32 50.2 4.9 1.88 0.42 % AUCextrap_pred 3.21 5.50 1.37 0.199.19 2.03 AUCinf_pred (mg · day/ml) 669 5 50.9 4.9 2.06 0.42AUCinf_pred/D (kg · day/ml) 0.223 0.002 0.170 0.016 0.0688 0.0140¹Half-life calculated on apparent beta phase ²Half-life calculated onapparent gamma phase

TABLE C-23 Basic pharmacokinetic parameters of NANOBODIES aftersubcutaneous administration in the female cynomolgus monkeys asdescribed in Example 14. RANKL008a RANKL001p RANKL003p parameter Mean SDMean SD Mean SD Vss/F (mL/kg)1 109 11 100 8 46.1 12.4 CL/F_pred(mL/day/kg) 7.93 0.70 11.7 0.2 6.66 0.65 MRTinf_pred (day)¹ 13.8 2.48.56 0.78 7.08 2.66 t 1/2 λz1 (day)² 10.9 2.1 5.81 0.28 5.25 0.23λz1_lower (day) — — — — — — λz1_upper (day) — — — — — — R2 t 1/2 λz1 — —— — — — t 1/2 λz2 (day)³ 2.07 0.99 1.86 0.28 1.03 0.37 λz2_lower (day) —— — — — — λz2_upper (day) — — — — — — R2 t 1/2 λz2 — — — — — — AUClast(mg · day/ml) 380 32 256 4 452 48 % AUCextrap_pred 0.005 0.002 0.1040.074 0.382 0.562 AUCinf_pred (mg · day/ml) 380 32 256 3 453 46.7 tmax(day) 2.00 0.00 2.00 0.00 3.00 2.65 Cmax (μg/ml) 21.8 1.7 24.6 0.5 46.211.0 AUCinf_pred/D (kg· day/ml) 0.127 0.011 0.085 0.001 0.151 0.016 F(%) 103%⁴ 74.0%⁴ 67.7%⁴ ¹MRT calculated as AUMC/AUC and therefore notcorrected for MAT. Vss/F = MRT · CL ²Half-life calculated on apparent bphase ³Half-life calculated on apparent g phase ⁴Estimation of F on the3 mg/kg dose level ignoring immunogenity and non-linearity in CL

TABLE C-24 Pharmacodynamic parameters (±SE) of RANK008a, RANKL001p andRANKL003p in the female cynomolgus monkey as described in Example 14.Parameter RANKL008a RANKL001p RANKL003p K_(in) (nM/day) 938 ± 194 1201 ±235   2835 ± 2758¹ K_(out) (1/day) 8.99 ± 1.79 11.7 ± 2.1   25.9 ± 19.8¹I_(max) 0.878 ± 0.024 0.882 ± 0.017 0.892 ± 0.038 IC₅₀ (μg/mL) 0.049 ±0.008 0.114 ± 0.025 0.518 ± 0.125 n 1.38 ± 0.29 1.23 ± 0.21 0.817 ±0.182 ¹Values could not be estimated with sufficient precision

TABLE C-25 Main pharmacokinetic parameters (mean +/− SD; n = 3) ofRANKL008a and αOPGL-1 following an single i.v. bolus administration withRANKL008a at 0.3 mg/kg or αOPGL-1 at 1 mg/kg in the female Cynomolgusmonkey. Compound RANKL008a αOPGL-1 Dose (mg/kg) 0.3 mg/kg 1 mg/kg RouteIV IV N n = 3 n = 2 C0 (ug/ml) 3.47 14.7 AUCinf (ug · d/ml) 22.8 94.5 t½(d) 8 6.5 Cl (ml/d/kg) 13.2 10.6 Vss (ml/kg) 77.1 72.8

TABLE C-26 Main average pharmacodynamic parameters (mean) of RANKL008aand αOPGL-1 following a single s.c. administration at 0.3 mg/kg(RANKL008a) or 1 mg/kg (αOPGL-1), respectively in the female Cynomolgusmonkey. Compound RANKL008a αOPGL-1 Dose (mg/kg) 0.3 1 Route IV IV N 3 2Tmin (d) 8 11 Rmin (%) 9 14 AUC below T (%*d) 289 212 Time below T (d)19 17.1 T onset (d) 0.143 0.125 T offset (d) 19.2 17.2

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

All references disclosed herein are incorporated by reference, inparticular for the teaching that is referenced hereinabove.

1.-61. (canceled)
 62. A method for the prevention and/or treatment of atleast one disease or disorder that is associated with RANK-L, with itsbiological or pharmacological activity, and/or with the biologicalpathways or signalling in which RANK-L is involved, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of at least one multivalent polypeptide,compound or construct, wherein the multivalent polypeptide, compound orconstruct comprises or essentially consists of at least two amino acidsequences and/or Nanobodies that specifically bind to RANKL, wherein theat least two amino acid sequences and/or Nanobodies essentially consistof 4 framework regions (FR1 to FR4 respectively) and 3 complementarydetermining regions (CDR1 to CDR3 respectively), in which: CDR1 ischosen from the group consisting of: a) the amino acid sequences of SEQID NOs: 188-249; b) amino acid sequences that have at least 80% aminoacid identity with at least one of the amino acid sequences of SEQ IDNOs: 188-249; or c) amino acid sequences that have 3, 2 or 1 amino aciddifference with at least one of the amino acid sequences of SEQ ID NOs:188-249; and/or CDR2 is chosen from the group consisting of: d) theamino acid sequences of SEQ ID NOs: 312-373 and 758; e) amino acidsequences that have at least 80% amino acid identity with at least oneof the amino acid sequences of SEQ ID NOs: 312-373 and 758; or f) aminoacid sequences that have 3, 2 or 1 amino acid difference with at leastone of the amino acid sequences of SEQ ID NOs: 312-373 and 758; and/orCDR3 is chosen from the group consisting of: g) the amino acid sequencesof SEQ ID NOs: 436-497; h) amino acid sequences that have at least 80%amino acid identity with at least one of the amino acid sequences of SEQID NOs: 436-497; or i) amino acid sequences that have 3, 2 or 1 aminoacid difference with at least one of the amino acid sequences of SEQ IDNOs: 436-497. 63.-66. (canceled)
 67. The method according to claim 62,wherein the at least two amino acid sequences and/or Nanobodiesessentially consist of 4 framework regions (FR1 to FR4 respectively) and3 complementary determining regions (CDR1 to CDR3 respectively), inwhich: CDR1 is chosen from the group consisting of: a) the amino acidsequences of SEQ ID NOs: 188-249; b) amino acid sequences that have atleast 80% amino acid identity with at least one of the amino acidsequences of SEQ ID NOs: 188-249; or c) amino acid sequences that have3, 2 or 1 amino acid difference with at least one of the amino acidsequences of SEQ ID NOs: 188-249; and CDR2 is chosen from the groupconsisting of: d) the amino acid sequences of SEQ ID NOs: 312-373 and758; e) amino acid sequences that have at least 80% amino acid identitywith at least one of the amino acid sequences of SEQ ID NOs: 312-373 and758; or f) amino acid sequences that have 3, 2 or 1 amino aciddifference with at least one of the amino acid sequences of SEQ ID NOs:312-373 and 758; and CDR3 is chosen from the group consisting of: g) theamino acid sequences of SEQ ID NOs: 436-497; h) amino acid sequencesthat have at least 80% amino acid identity with at least one of theamino acid sequences of SEQ ID NOs: 436-497; or i) amino acid sequencesthat have 3, 2 or 1 amino acid difference with at least one of the aminoacid sequences of SEQ ID NOs: 436-497.
 68. The method according to claim62, wherein the at least two amino acid sequences and/or Nanobodiesessentially consist of 4 framework regions (FR1 to FR4 respectively) and3 complementary determining regions (CDR1 to CDR3 respectively), inwhich the CDR sequences of said amino acid sequences and/or Nanobodieshave at least 70% amino acid identity, preferably at least 80% aminoacid identity, more preferably at least 90% amino acid identity, such as95% amino acid identity or more or even essentially 100% amino acididentity with the CDR sequences of at least one of the amino acidsequences of SEQ ID NOs: 560-621.
 69. The method according to claim 62,wherein the at least two amino acid sequences essentially consist of adomain antibody (or an amino acid sequence that is suitable for use as adomain antibody), of a single domain antibody (or an amino acid sequencethat is suitable for use as a single domain antibody), of a “dAb” (or anamino acid sequence that is suitable for use as a dAb), a Nanobody(including but not limited to a V_(HH) sequence), a partially humanizedNanobody or a fully humanized Nanobody.
 70. The method according toclaim 62, wherein the at least two amino acid sequences and/orNanobodies are a V_(HH) sequence, a partially humanized V_(HH) sequence,a fully humanized V_(HH) sequence, a camelized heavy chain variabledomain or a Nanobody that has been obtained by affinity maturation. 71.The method according to claim 62, wherein the at least two amino acidsequences essentially consist of a Nanobody that i) has at least 80%amino acid identity with at least one of the amino acid sequences chosenfrom the group consisting of SEQ ID NOs: 1-22, 560-621, 730-757 and 765,in which for the purposes of determining the degree of amino acididentity, the amino acid residues that form the CDR sequences aredisregarded; and in which: ii) preferably one or more of the amino acidresidues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108according to the Kabat numbering are chosen from the Hallmark residuesmentioned in Table A-3.
 72. The method according to claim 62, whereinthe multivalent polypeptide, compound or construct comprises at leasttwo Nanobodies that are chosen from the group consisting of SEQ ID NOs:560-621 or SEQ ID NOs: 730-757 and 765 or from the group consisting ofamino acid sequences that have more than 80%, preferably more than 90%,more preferably more than 95%, such as 99% or more sequence identity (asdefined herein) with at least one of the amino acid sequences of SEQ IDNOs: 560-621 or SEQ ID NOs: 730-757 and
 765. 73. The method according toclaim 62, wherein the multivalent polypeptide, compound or construct isa bivalent polypeptide, compound or construct.
 74. The method accordingto claim 62, wherein the multivalent polypeptide, compound or constructcomprises or essentially consists of an amino acid sequence chosen fromSEQ ID NOs: 622-729, 759-762 and 766-773.
 75. The method according toclaim 62, wherein the multivalent polypeptide, compound or constructessentially consists of a polypeptide that i) has at least 80% aminoacid identity with at least one of the amino acid sequences of SEQ IDNOs: 622-729, 759-762 and 766-773, in which for the purposes ofdetermining the degree of amino acid identity, the amino acid residuesthat form the CDR sequences are disregarded; and in which: ii)preferably one or more of the amino acid residues at positions 11, 37,44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numberingare chosen from the Hallmark residues mentioned in Table A-3.
 76. Themethod according to claim 62, wherein the multivalent polypeptide,compound or construct is a multispecific construct.
 77. The methodaccording to claim 62, wherein the multivalent polypeptide, compound orconstruct further comprises one or more other groups, residues, moietiesor binding units, optionally linked via one or more linkers.
 78. Themethod according to claim 77, in which said one or more other groups,residues, moieties or binding units provide the multivalent polypeptide,compound or construct with increased half-life.
 79. The method accordingto claim 78, in which said one or more other groups, residues, moietiesor binding units that provide the multivalent polypeptide, compound orconstruct with increased half-life is chosen from the group consistingof serum proteins or fragments thereof, binding units that can bind toserum proteins, an Fc portion, and small proteins or peptides that canbind to serum proteins.
 80. The method according to claim 78, in whichsaid one or more other groups, residues, moieties or binding units thatprovides the multivalent polypeptide, compound or construct withincreased half-life are chosen from the group consisting of bindingunits that can bind to serum albumin (such as human serum albumin) or aserum immunoglobulin (such as IgG).
 81. The method according to claim78, wherein said one or more other groups, residues, moieties or bindingunits that provides the multivalent polypeptide, compound or constructwith increased half-life are chosen from the group consisting of domainantibodies, amino acid sequences that are suitable for use as a domainantibody, single domain antibodies, amino acid sequences that aresuitable for use as a single domain antibody, “dAb”'s, amino acidsequences that are suitable for use as a dAb, or Nanobodies that canbind to serum albumin (such as human serum albumin) or a serumimmunoglobulin (such as IgG).
 82. The method according to claim 78, inwhich said one or more other binding units that provides the multivalentpolypeptide, compound or construct with increased half-life is chosenfrom SEQ ID NOs: 790-791.
 83. The method according to claim 62, whereinthe at least one disease or disorder that is associated with RANK-L,with its biological or pharmacological activity, and/or with thebiological pathways or signalling in which RANK-L is involved, is a bonedisease or disorder.
 84. The method according to claim 83, wherein saidbone disease or disorder is chosen from the group consisting ofosteoporosis, Paget's disease, osteomyelitis, hypercalcemia,osteonecrosis, osteopenic disorders, arthritic disorders andperiprosthetic osteolysis.
 85. A multivalent polypeptide, compound orconstruct comprising or essentially consisting of at least two aminoacid sequences and/or Nanobodies that are directed against and/or thatcan specifically bind to RANK-L, in which the CDR sequences of saidamino acid sequences and/or Nanobodies have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof the amino acid sequence of SEQ ID NOs: 560-621 and SEQ ID NOs:730-757 and 765.